DETERMINING THE SPECIFICITY OF PEPSIN FOR PROTEOLYTIC DIGESTION A thesis presented by Melissa H. Palashoff to The Department of Chemistry and Chemical Biology in partial fulfillment of the requirements for the degree of Master of Science in the field of Chemistry Northeastern University Boston, Massachusetts August 2008 1
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DETERMINING THE SPECIFICITY OF PEPSIN
FOR PROTEOLYTIC DIGESTION
A thesis presented
by
Melissa H. Palashoff
to The Department of Chemistry and Chemical Biology
in partial fulfillment of the requirements for the degree of Master of Science
in the field of
Chemistry
Northeastern University Boston, Massachusetts
August 2008
1
DETERMINING THE SPECIFICITY OF PEPSIN
FOR PROTEOLYTIC DIGESTION
by
Melissa H. Palashoff
ABSTRACT OF THESIS
Submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemistry and Chemical Biology
in the Graduate School of Arts and Sciences of Northeastern University, August 2008
2
ABSTRACT
Pepsin is an aspartic acid protease that is commonly found in the stomach of
many organisms. Porcine pepsin is the most studied and is fully active at pH 1.9 but
inactive above pH ~7. Pepsin is known to have limited specificity and there are only
general rules about its cleavage preferences.
To further define rules regarding pepsin specificity, a database was constructed
consisting of 40 proteins and 1344 peptide cleavages from the literature. Contemporary
scientific literature was searched for all publications that involve pepsin digestion and
mass spectrometry at pH 2.5-2.7. Peptide data for 40 proteins were extracted and
combined to create a map of pepsin cleavage specificity. The frequency of cleavage for
each protein was normalized based on how many times that specific combination of
residues occurred in the protein sequence.
In addition to the literature search, nine proteins along with E.coli whole cell
lysate were digested at pH 1.0, 2.5 and 4.0. The proteins were analyzed with online
pepsin digestion using an immobilized pepsin column and UPLC/ESI-MSE. The peptides
and their fragments were identified with a combination of MSE, software analysis, and
manual inspection.
The analysis of the data indicated that pepsin maintains limited cleavage
preferences. At pH 2.5, pepsin will cleave preferentially after most bulky, hydrophobic
amino acids such as leucine and phenylalanine. Additionally, the residues that most often
occur immediately following the cleaved peptide bond are tryptophan and tyrosine. It has
also been shown that pepsin will rarely cleave at proline and histidine. Analysis
performed at pH 1.0 and 4.0 yielded similar results.
3
ACKNOLEDGEMENTS
I would like to begin by thanking my advisor Dr. John R. Engen for all of his help
in making me a better scientist. Without his guidance and advice this research would not
have been possible. I am also grateful to my other committee members, Dr. Mary Jo
Ondrechen and Dr. Paul Vouros, for helping to make this thesis the best that it could be.
I would like to sincerely thank everyone in Dr. Engen’s lab for their continuous
support over the past year. To my labmates, Dr. Thomas Wales, Dr. Roxana Iacob,
Christopher Morgan, Sean Marcsisin, Damian Houde and Susan Fang, for providing a
wonderful environment for me to work and learn in.
Finally, I would like to dedicate this to my family and friends for their constant
encouragement during the past five years. I would especially like to thank my parents,
William and Patricia, and my brother Joshua for everything they have done to support
me, both morally and materially, during my college career. Last, but certainly not least,
to Eddie for always being there to support me during the good days and the bad, thank
you.
4
TABLE OF CONTENTS
ABSTRACT ……………………………………………………………………………………….
TABLE OF CONTENTS ………………………………………………………………………….
LIST OF FIGURES ………………………………………………………………………………..
LIST OF TABLES ………………………………………………………………………………...
LIST OF ABBREVIATIONS ……………………………………………………………………
CHAPTER ONE: INTRODUCTION AND BACKGROUND TO ASPARTIC ACID
The aspartic acid protease family can be split up into five main categories. Pepsin-like contains 70 members, retroviral contains 45 members, cauliflower mosaic contains 6,
t i l t i 1 d i t t i 6 O l l ti fspumaretroviral contains 1, and copia transposon contains 6. Only a selection of enzymes are shown in this diagram. Adapted from Dunn (2001) and Rawlings, Morton et al. (2008).
14
1.2 Catalytic Mechanism of Aspartic Proteases
As previously mentioned, aspartic proteases contain a conserved segment of
residues. This amounts to about a 5% sequence identity in all of the enzymes. Two of
the residues that remain conserved are aspartic acids (hence the name aspartic acid
proteases). The aspartic residues, Asp32 and Asp215 (pepsin numbering), are located in
the active site cleft of the enzyme and are involved in the catalytic mechanism (Dunn
1989). To date the most popular mechanism of aspartic proteases (Figure 1.2) was
proposed by Northrop (Northrop 2001) and is believed to involve the controversial low-
barrier hydrogen bond.
The mechanism begins with the free enzyme wherein the two aspartate groups are
believed to form a 10-atom cyclic structure with a water molecule. This forms a loose
complex with the substrate in step 1. The catalytic process begins in step 2 when the
complex takes on the required geometry and distances. In step 3 a proton is removed
from the bound water molecule in order to generate a hydroxide ion, which attacks the
carbon atom of the substrate’s carboxyl group. A proton is then transferred to the
nitrogen of the peptide bond in step 4 and bond cleavage occurs in step 5. In step 6 the
complex opens up for release of the products in step 7. A proton is then lost in step 8 and
finally a new water molecule is bound to the complex in step 9. Steps 8 and 9 represent
the deprotonation, rehydration and restructuring of the complex to complete
isomerization. Isomerization is necessary in order to reform the low-barrier hydrogen
bond (Northrop 2001; Dunn 2002).
15
1 2 3
4 5 6
7 8 9
Figure 1.2. The aspartic protease catalytic mechanism. The first step in the mechanism is the formation of a loose complex upon substrate binding. Step 2 begins the catalytic process and in step 3 the carbonyl group is attacked following the removal of a proton. In step 4 there is the transfer of a proton and in step 5 bond cleavage occurs. The complex opens up in step 6 and the product is released in step 7. In step 8 a proton is lost and in step 9 a new water molec le binds to the comple Adapted from Northrop (2001)molecule binds to the complex. Adapted from Northrop (2001).
16
1.3 Primary Structure of the Pepsin-like Family
Pepsin-like proteases make up the largest family of acid proteases. They are
roughly 330-350 residues in length and share a common primary structure. Pepsin-like
proteases are single chain enzymes that are usually described as having two domains.
The primary structure is evidence as to why the protein is described as having two-
domains as it has a repetitive form (Orengo, Michie et al. 1997).
The sequence typically begins with 30 to 35 residues followed by two
hydrophobic residues (Hpb). The hydrophobic residues are followed by the conserved
Asp-Thr-Gly sequence found in all aspartic proteases. The Asp-Thr-Gly sequence occurs
within a wide loop known as the Psi-loop. About 45 residues after the Asp-Thr-Gly
sequence a conserved Tyr occurs. After about another 45 residues there is a Leu-Gly-Ile
sequence followed by about 90 more residues. The sequence then shows some form of
repetition. Two hydrophobic residues occur again before the Asp-Thr-Gly sequence.
There are about 85 more residues before a Leu-Gly-Asp sequence. 20 to 30 more amino
acids make up the rest of the primary structure of a pepsin-like protease (Davies 1990).
The tyrosine residue that is conserved in the sequence is important because it
helps to define the active site pockets where substrate side-chain residues bind. In the
Leu-Gly-Ile and Leu-Gly-Asp sequence the glycine occurs for structural reasons as it is
easily able to fit through the Psi-loop. The leucine, isoleucine and aspartic acid residues
are there because their bulkiness ensures that the Psi-loop is locked into place (Dunn
Figure 1.3. Sequence alignment of porcine pepsinogen and porcine pepsin. The 44 residue propeptide is highlighted in blue. This segment is cleaved off upon activation.
19
inhibits the activity of the enzyme because a segment of it blocks access to the catalytic
aspartates in the active site. Removal of the propeptide results in the activation of
pepsinogen to pepsin (James and Sielecki 1986). A loss of helical structure of the
propeptide also typically occurs during activation of the zymogen (Davies 1990).
Pepsinogen activation occurs when the pH of a solution of pepsinogen is lowered.
The lowering of the pH is believed to protonate the carboxylate side chains of pepsin
which causes the complex to break down and leads to the formation of the active enzyme.
Raising the pH can fully reverse the zymogen activation if performed in a timely manner.
However, if the pH is lowered for a prolonged period of time the activation is irreversible
(James and Sielecki 1986).
The activation of pepsinogen into pepsin is believed to occur through two
pathways, either in a one-step process or in a sequential manner. There are also two
different reactions that occur during activation. In the intramolecular reaction pepsinogen
cleaves itself to form the active pepsin, while in the intermolecular reaction pepsinogen is
cleaved by either another pepsinogen molecule, an intermediate form or an active pepsin
molecule. Kinetic experiments have shown that the intramolecular reaction is
predominant at a pH lower than 3.0 (al-Janabi, Hartsuck et al. 1972). The one-step
activation pathway appears to proceed mainly, but not exclusively, through the
intermolecular reaction (Kageyama and Takahashi 1983).
Both the one-step pathway and the stepwise pathway are believed to occur
simultaneously during the activation of pepsinogen to pepsin (Christensen, Pedersen et al.
1977). The intramolecular reaction and the intermolecular reaction are both involved in
the one-step pathway. It appears as though the intramolecular reaction is an essential part
20
for the initial activation in order to generate the active pepsin molecules. The
intermolecular reaction is important for completion of the activation (Kageyama and
Takahashi 1987).
1.4.3 Pepsin Crystal Structure
Porcine pepsin was first crystallized in 1930 by John Northrop and later refined
by Sielecki et al. in 1990 (Sielecki, Fedorov et al. 1990). Figure 1.4 illustrates the crystal
structure of human pepsin (Fujinaga, Chernaia et al. 1995). The catalytic Asp residues,
Asp32 and Asp215, are highlighted in blue while the pepsin inhibitor pepstatin is
highlighted in red. The protein can be divided up into three regions (James and Sielecki
1986). The first region consists of a six-stranded antiparallel β-sheet. This interdomain
forms the backbone of the structure and is located behind the catalytic site region. The
other two domains consist of two lobes. One lobe is the N-terminal which consists of
142 residues and the other lobe is the C-terminal which consists of 123 residues. Despite
a similar pattern in their amino acid sequences, the N-terminal and C-terminal domains
are not very similar in their secondary or tertiary structures (Sielecki, Fedorov et al.
1990).
Other elements of the pepsin crystal structure are that it consists of a short
interdomain peptide that is next to the external side of the six-stranded β-sheet (Sielecki,
Fedorov et al. 1990). There are also two strands that form a β-hairpin loop that is often
called the flap. The flap projects out at the active site cleft of the molecule (Davies
1990). Pepsin contains a large hydrophobic core at its center. This is a result of the
reassembly of three regions mentioned above. A major factor contributing to the
21
Asp215
Asp32
Figure 1.4. Crystal structure of human pepsin. The two catalytic Asp residues, Asp32 and Asp215, are highlighted in blue. The pepsin inhibitor pepstatin is highlighted in red. PDB: 1PSN
22
hydrophobic core are side chains that protrude inward from the six-stranded β-sheet
(Sielecki, Fedorov et al. 1990).
The catalytic site of pepsin is highlighted by two aspartic acid residues, Asp32 and
Asp215. There is an Asp residue located in both the N-terminal and C-terminal domain.
The two Asp residues are located towards the end of each domain and are connected
through a network of hydrogen bonds. The active site is quite rigid. However, the flap
that protrudes out above the active site is rather flexible. The flap can close around
inhibitors that are bound to the active site, thus limiting the mobility of the flap (James,
Sielecki et al. 1982).
1.4.4 Activity of Pepsin
Pepsin is an enzyme whose activity is greatly dependent on its pH. Pepsin has its
optimum enzymatic activity at a pH between 1.8 and 2.0. It is remains stable, and still
highly active, when the pH drops to as low as 1.0 (Ryle 1970). Pepsin will begin to lose
activity around pH 5 (Smith 1991) and it becomes irreversibly inactive at a pH around 7.
However, a high concentration of pepsin will not become inactive until a pH of about 8
(Jones and Landon 2002). The activity of pepsin is also dependent upon the enzyme to
protein ratio. The higher this ratio is the more efficient the enzyme becomes (Wu, Kaveti
et al. 2006).
1.4.5 Pepsin and Proteomics
Pepsin can be a very useful tool for proteomics. Pepsin has a very broad
specificity and is believed to often cleave after bulky hydrophobic residues (Fruton 1970;
23
Ryle 1970). Because of its broad specificity pepsin produces many peptides during
digestion. The multiple cleavage sites means that the peptides produced are usually
small, around 3 to 30 residues in length. The peptides are also typically overlapping
which is useful for protein mapping. Despite its broad specificity pepsin is still a very
reproducible enzyme, meaning it will yield the same peptides when digestion of a protein
is performed at identical conditions (Zhang and Smith 1993).
1.5 Research Objectives
As previously mentioned there is little known about its specificity other than the
fact that it prefers to cleave after bulky hydrophobic residues (Fruton 1970). Determining
trends in pepsin specificity is important because pepsin is an enzyme that is widely used
and without any rules about its cleavage preferences, protein characterization can be very
difficult. The reason why pepsin is used for protein characterization despite the fact it
has little known specificity is because it is one of the only enzymes to have a high
activity in the low pH range.
One of the most common uses for pepsin is when performing hydrogen /
deuterium exchange mass spectrometry (HXMS). To date, pepsin is essentially the only
enzyme that can be used for these experiments. Due to the nature of the reaction, the
digests need to be performed at a pH around 2.5 and at 0 °C. While there are other
aspartic proteases that will work at this pH requirement there are no other enzymes that
remain as active as pepsin is at this low temperature. Since pepsin is so often used to
characterize proteins and because there is so little known about pepsin cleavage
preferences, the main objective of this research is to determine if pepsin has any
24
specificity, thus advancing the use of pepsin digestion for protein characterization by
mass spectrometry.
The steps taken to determine trends in pepsin specificity consist of two main
parts. Chapter 2 outlines an extensive literature search performed in order to gather all
existing data involving pepsin cleavages. The corresponding experimental research is
discussed in chapter 3. The goal of the experimental research was to gain more data of
pepsin digests. These analyses were performed at pH 2.5 which is the most common pH
for pepsin digests. Another aspect of the experiment which is discussed in chapter 3 is
the determination of the effect of pH on the specificity of pepsin. It is known that
pepsin’s activity is highly dependent on pH (Ryle 1970) but it is not known whether or
not that loss or gain of activity is directly related to specificity.
1.6 References
al-Janabi, J., J. A. Hartsuck, et al. (1972). "Kinetics and mechanism of pepsinogen
activation." J Biol Chem 247: 4628-32.
Bernal, J. D. and D. Crowfoot (1934). "X-Ray photographs of crystalline pepsin." Nature
133: 794-795.
Christensen, K. A., V. B. Pedersen, et al. (1977). "Identification of an enzymatically
active intermediate in the activation of porcine pepsinogen." FEBS Lett 76: 214-8.
Davies, D. R. (1990). "The structure and function of the aspartic proteinases." Annu Rev
Biophys Biophys Chem 19: 189-215.
25
Dickson, C., Eisenman, R., Fan, H., Hunter, E., Teich, N. (1984). RNA Tumor Viruses.
R. Weiss, Teich, N., Varmus, H., Coffin, J. New York, Cold Spring Harbor Lab:
513-648.
Dunn, B. M. (1989). Determination of Protease Mechanism. Proteolytic Enzymes: A
Practical Approach. R. J. a. B. Beynon, J. S. Oxford, England, Information Press
Ltd.: 57-81.
Dunn, B. M. (2001). "Overview of pepsin-like aspartic peptidases." Curr Protoc Protein
Sci Chapter 21: Unit 21 3.
Dunn, B. M. (2002). "Structure and mechanism of the pepsin-like family of aspartic
peptidases." Chem Rev 102: 4431-58.
Fruton, J. S. (1970). "The specificity and mechanism of pepsin action." Adv Enzymol
Relat Areas Mol Biol 33: 401-43.
Fruton, J. S. (1976). "The mechanism of the catalytic action of pepsin and related acid
proteinases." Adv Enzymol Relat Areas Mol Biol 44: 1-36.
Fujinaga, M., M. M. Chernaia, et al. (1995). "Crystal structure of human pepsin and its
complex with pepstatin." Protein Sci 4: 960-72.
Gillespie, A. L. (1898). The Natural History of Digestion. London, W. Scott.
James, M. N., A. Sielecki, et al. (1982). "Conformational flexibility in the active sites of
aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin."
Proc Natl Acad Sci U S A 79: 6137-41.
James, M. N. and A. R. Sielecki (1986). "Molecular structure of an aspartic proteinase
zymogen, porcine pepsinogen, at 1.8 A resolution." Nature 319(6048): 33-8.
26
Jones, R. G. and J. Landon (2002). "Enhanced pepsin digestion: a novel process for
purifying antibody F(ab')(2) fragments in high yield from serum." J Immunol
Methods 263: 57-74.
Kageyama, T. and K. Takahashi (1983). "Occurrence of two different pathways in the
activation of porcine pepsinogen to pepsin." J Biochem 93: 743-54.
Kageyama, T. and K. Takahashi (1987). "Activation mechanism of monkey and porcine
pepsinogens A. One-step and stepwise activation pathways and their relation to
intramolecular and intermolecular reactions." Eur J Biochem 165: 483-90.
Northrop, D. B. (2001). "Follow the protons: a low-barrier hydrogen bond unifies the
mechanisms of the aspartic proteases." Acc Chem Res 34: 790-7.
Northrop, J. H. (1930). "Crystalline pepsin I. Isolation and tests for purity." J. Gen.
Physiol. 13: 739-766.
Orengo, C. A., A. D. Michie, et al. (1997). "CATH--a hierarchic classification of protein
domain structures." Structure 5: 1093-108.
Perlmann, G. E. (1963). "The optical rotatory properties of pepsinogen." J Mol Biol 6:
452-64.
Rawlings, N. D., F. R. Morton, et al. (2008). "MEROPS: the peptidase database." Nucleic
Acids Res 36: D320-5.
Ryle, A. (1970). "The Porcine Pepsin and Pepsinogens." Methods Enzymol 19: 316-336.
Sielecki, A. R., A. A. Fedorov, et al. (1990). "Molecular and crystal structures of
monoclinic porcine pepsin refined at 1.8 A resolution." J Mol Biol 214: 143-70.
27
Smith, J. L., Billings, G. E., Yada, R. Y (1991). "Chemical Modification of Amino
Groups in Mucor miehei Aspartyl Proteinase, Porcine Pepsin, and Chymosin. I.
Structure and Function." Agricultural and Biological Chemistry 55: 2009-2016.
Sorensen, S. P. L. (1909). "Enzymstudien II. Mitteilung. Uber die Messung und die
Bedeutung der Wasserstoffionen-konzentration bei enzymatischen Prozessen."
Biochem. Z. 21: 201-304.
Wu, Y., S. Kaveti, et al. (2006). "Extensive deuterium back-exchange in certain
immobilized pepsin columns used for H/D exchange mass spectrometry." Anal
Chem 78: 1719-23.
Zhang, Z. and D. L. Smith (1993). "Determination of amide hydrogen exchange by mass
spectrometry: a new tool for protein structure elucidation." Protein Sci 2: 522-31.
28
CHAPTER 2
LITERATURE SEARCH
2.1. Introduction
The first part of this research project was to conduct a search of contemporary
scientific literature for publications that involve pepsin digestion and mass spectrometry.
Performing this literature search was useful because it provided a large amount of
cleavages from a diverse set of proteins. This extensive amount of data is a very good
basis for determining pepsin specificity.
A literature analysis of pepsin specificity has been performed previously (Keil
1992). However, the search performed in this book was a very broad one. There were no
limitations put on the pH at which the digestions were performed. As previously
mentioned pepsin can be greatly affected by pH. Since this search takes into account
digestions performed at a wide range of pH values, some of the specificity results could
be skewed because of it.
The literature search performed for this project puts strict limitations on the pH at
which the digestions could be performed. In order to be considered in this literature
search digestion needed to be performed within a pH range of 2.5 to 2.7. Pepsin
experiences a very high activity within these values. This pH range is also of
considerable importance because it is the range at which HXMS experiments are
conducted. As previously mentioned HXMS experiments are one of the main types of
experiments that utilize pepsin digestions. Due to this fact, there is a vast amount of
literature published containing digestions within these pH values.
29
2.2. Materials and Methods
Search engines for online databases were used to conduct the literature search.
As mentioned above the literature that was of interest involved pepsin digestions and
mass spectrometry. The initial search yielded hundreds of results. The results were
narrowed by only choosing digestions that were performed within a pH range of 2.5 to
2.7.
Once the results were limited to those containing digests performed at pH 2.5 to
2.7, all of the papers were scanned to see if they contained a peptic digest map (Figure
2.1). If two or more sources used the same peptic digest map only one was used to
extract data from. Likewise, if two or more sources contained digest maps of the same
protein from the same organism the most comprehensive map was used ensuring that a
specific protein only be considered once when gathering data. The final results yielded
peptic digest maps from 40 sources. Table 2.1 lists the references along with the
protein(s) studied in the publication.
2.3. Construction of Cleavage Database
All of the peptic digest maps retrieved from the literature search were analyzed in
order to create a database of pepsin cleavages. The database consists of the residues that
are found in the P4 to P4’ positions (see table 2.2 at end of chapter). As shown in Figure
2.2, residues P1 through P4 occur before the cleavage and residues P1’ through P4’ occur
after the cleavage. The database incorporates out to the P4 and P4’ position because there
has been some evidence that residues in these positions can effect pepsin specificity (Keil
Figure 2.1. Example of a peptic digest map. Peptic peptides are underlined in red. Pepsin will produce multiple overlapping peptides.
31
Table 2.1. Literature search results Reference Protein
Lu, Wintrode et al. 2007 Major prion protein (human) Man, Montagner et al. 2007 Myoglobin (sperm whale) Brier, Maria et al. 2007 CENP-E (human) Cheng, Cusanovich et al. 2006 Photoactive yellow protein Cheng, Wysocki et al. 2006 cytochrome c2 (rhodobacter capsulatus) Hochrein, Wales et al. 2006 HIV and SIV Nef Shi, Koeppe et al. 2006 AChBP from L. stagnalis Tsutsui, Liu et al. 2006 α1AT (human) Wales and Engen 2006 Lyn SH3 and α-spectrin SH3 Weis, Kjellen et al. 2006 Lck SH3 Yao, Zhou et al. 2006 Cks1 and Skp2 Catalina, Fischer et al. 2005 SH2 domains of Syk tSH2 Kang and Prevelige 2005 P22 capsid coat protein Lee, Hoofnagle et al. 2005 ERK2 Brier, Lemaire et al. 2004 Kinesin-like protein KIF11 (human) Casbarra, Birolo et al. 2004 Human α-LA Croy, Koeppe et al. 2004 human and bovine α-thrombin Croy, Bergqvist et al. 2004 IκBα Li, Chou et al. 2004 LR3IGF-I Mazon, Marcillat et al. 2004 Creatine kinase M-type (rabbit) Wu, Hasan et al. 2004 human rCRALBP Yan, Broderick et al. 2004 human RXRα LBD Anand, Law et al. 2003 PKA Chik and Schriemer 2003 rabbit muscle actin Cravello, Lascoux et al. 2003 PBP-2X* Rist, Jorgensen et al. 2003 σ 32 Wintrode, Friedrich et al. 2003 HSP16.9 Hasan, Smith et al. 2002 α-Crystallin (αA and αB) Yan, Zhang et al. 2002 rhM-CSFß Hughes, Mandell et al. 2001 CheB Wang, Lane et al. 2001 BMV Chen and Smith 2000 GroEL Engen, Smithgall et al. 1999 Hck SH(3 + 2) Wang, Li et al. 1999 cNTnC Resing and Ahn 1998 human MKK1 Neubert, Walsh et al. 1997 recoverin Wang, Blanchard et al. 1997 DHPR Dharmasiri and Smith 1996 horse heart cyt c Zhang, Post et al. 1996 rabbit muscle aldolase Johnson and Walsh 1994 equine myoglobin
32
P P P P P ’ P ’ P ’ P ’P4 P3 P2 P1 P1 P2 P3 P4 T E E D A A S Y
RRGAISAEVY TEEDAASYIR KVIPKDYKTM
AALAKAIEKN VLFSHLDDNE RSDIFDAMFP
VSFIAGETVI QQGDEGDNFY VIDQGEMDVY
Figure 2.2. Example of cleavage nomenclature. The P4 through P1 residues are those that occur before the cleavage site. The P1’ through P4’ residues occur after the cleavage site.
33
After the database was constructed the residues in the P1 and P1’ positions were
focused on as these are believed to have the most influence on pepsin specificity
(Hamuro, Coales et al. 2008). For the analysis, all of the cleavages between two specific
residues were tallied to construct a matrix of cleavage data (Table 2.3).
As shown in Table 2.3 the residue that occurred most often before the cleavage
site (the P1 position) is leucine. Out of the 1,344 cleavages 372, or 28%, occurred after a
leucine. The residues that occurred most often in the P1’ position are leucine and alanine.
The residues that occur least often before the cleavage point are proline, histidine and
lysine. Glycine, proline and lysine are the three residues that produce the least number of
cleavages when found in the position following the cleavage site. This matrix of
cleavage data is comprehensive of the literature search performed; however there are
some issues with it. It is difficult to extract any trends in pepsin specificity just by
looking at the table. Another problem is that this raw data does not take into account the
abundance of amino acids. To make the values more meaningful a normalization of the
data must be performed.
2.4. Data Normalization
Normalizing the data is important because it takes into account how often a
specific amino acid occurs in a protein’s sequence. Taking the data in Table 2.3 for
example, 36 cleavages occurred between a leucine and a leucine while only three
cleavages occurred between a two tryptophans. When looking at the sequences for the
proteins in the literature there are 85 times in which two leucines occur next to one
another, thus pepsin produced a cleavage between a leucine and a leucine 36 out of 85
34
Table 2.3. Sum of cleavages betw
een two residues
P1 '
A
C
D
E
F G
H
I
K
L M
N
P
Q
R
S T
V W
Y A
5
1 2
6 10
0 1
164
14 2
1 1
1 2
3 2
110
12C
1
1 1
1 0
0 0
1 0
2 1
0 2
0 0
1 1
3 0
1 D
6
1 3
4 6
1 0
140
7 1
3 4
2 2
1 2
9 3
6 E
11 1
115
7 1
2 10
6 20
6 6
2 4
101
2 16
3 10
F 17
2 13
9 10
9 3
118
14 3
6 2
5 10
115
152
10G
4
2 0
3 4
3 1
4 1
5 2
1 0
1 2
3 2
3 2
4 H
1
0 1
0 0
0 0
0 0
1 0
0 0
0 0
0 0
0 0
0 I
5 0
0 1
5 0
0 2
1 5
1 2
0 1
2 3
3 3
0 1
K
3 0
2 4
0 0
0 1
2 1
0 2
0 0
0 1
3 1
0 1
P1
L 39
9 25
2819
1313
1714
36 12
158
1415
2222
259
17M
7
1 3
106
2 1
4 9
4 4
2 0
0 0
5 1
7 2
3 N
0
1 2
1 6
2 0
5 1
2 2
1 1
0 1
1 3
7 1
4 P
0 0
1 0
0 0
0 1
0 0
0 1
0 0
0 0
1 1
0 0
Q
3 0
3 2
3 0
0 5
1 4
0 1
0 1
1 0
1 7
1 5
R
0 0
1 2
1 0
0 1
1 3
0 0
1 1
1 1
2 2
2 0
S 4
0 2
3 4
4 1
4 2
6 2
0 2
0 1
0 0
6 3
8 T
6 0
4 1
6 5
0 6
0 7
1 1
0 1
4 0
0 2
1 6
V 5
0 4
1 2
1 0
2 3
3 1
0 0
1 1
3 3
2 1
5 W
0
0 1
0 3
0 1
2 0
3 0
0 0
2 0
0 0
0 3
2 Y
3 1
0 1
4 2
0 5
1 4
2 3
0 2
1 2
2 11
2 4
Tally of cleavages between tw
o specific residues. Residues in the P
1 positions are in the left hand column and residues in the P
1 ’ positions are along the top row
. For example, cleavage betw
een a phenylalanine and glycine occurred 9 times w
hile cleavage between
a leucine and a leucine occurred 36 times.
35
possible times. Two tryptophans occurred next to each other in the primary structures
only five times. Therefore, pepsin produced a cleavage between a tryptophan and a
tryptophan three out of five times. Leucine is one of the more abundant amino acids
while tryptophan is not. This is why data normalization is needed.
The first step to normalizing the raw data was to gather the sequences of all of the
proteins from the literature search. A matrix of possible cleavages was then constructed
(Table 2.4). The total number of possible cleavages for this set of proteins is 11,240.
The data was then normalized using the equation found in Figure 2.3 (Keil 1992). The
normalized values are shown in Table 2.5.
The matrix in Table 2.5 was then transformed into cleavage data map. The cleavage
data map is a more illustrative way to represent the data than the matrix form. It is much
easier to decipher trends in pepsin specificity by glancing at the cleavage map than it is to
look at the matrix. The cleavage data map is shown in Figure 2.4.
2.5. Cleavage Data Map
The cleavage data map in Figure 2.4 shows some trends in pepsin specificity. On
the y-axis are the P1 residues and on the x-axis are the P1’ residues. The size of the
square is the normalized cleavage value. For example, the probability that pepsin will
cleave between leucine and tryptophan is 7.5 while the probability that pepsin will cleave
between phenylalanine and glycine is 1.8. The map is arranged so that the residues
where pepsin prefers to cleave most often are nearest to the zero point on the chart and
those residues where pepsin does not prefer to cleave are farther out.
36
Table 2.4. Possible cleavages between tw
o residues P
1 '
A
C
D
E F
G
H
I K
L
M
N
P Q
R
S
T V
W
Y A
89
7 55
5624
7318
6155
75 22
3235
3834
4745
559
24C
8
1 13
7 7
123
7 7
14 4
5 8
105
128
9 0
4 D
47
8 47
4927
6012
4742
75 16
2127
1934
3818
449
21E
66 6
5590
3158
2148
7879
2434
19 33
4729
4950
1023
F 34
6 39
2122
4211
2431
33 6
2517
1824
3223
232
15G
56
11 47
5835
5814
4958
61 27
3422
3642
5159
5911
34H
12
4 10
1812
2544
6 18
30 13
6 21
8 14
2214
171
15I
47 8
3327
2431
2332
3761
1216
30 25
3644
4545
8 20
K
77 11
4860
2757
2739
6868
1632
37 24
3035
3456
1125
P1
L 92
16 58
8838
6128
3473
85 18
5039
5453
8270
5110
24M
25
2 19
2714
236
1228
29 7
6 11
7 13
1815
212
7 N
20
7 19
3620
268
2730
48 16
1522
1419
2924
346
14P
30 7
3456
2836
1423
3039
1216
16 12
2045
2433
1111
Q
27 3
2331
1130
1028
3247
1715
22 19
2030
2139
3 9
R
40 10
3447
2427
1234
3245
9 18
35 23
3135
3540
8 14
S 40
12 43
4831
5924
3538
65 12
2746
2937
5731
439
23T
51 5
2751
3051
1533
3868
1721
34 22
2930
4238
9 19
V 64
14 37
5731
4416
3757
69 18
4033
2138
5137
406
17W
8
2 6
6 5
113
9 11
10 1
7 2
107
6 7
7 5
7 Y
18 5
2017
7 23
4 20
2229
9 18
21 17
2219
2326
3 9
Tally of possible cleavages between tw
o specific residues. Residues in the P
1 positions are in the left hand column and residues in the
P1 ’ positions are along the top row
. For example, cleavage betw
een a phenylalanine and glycine could occur 42 times w
hile cleavage betw
een a leucine and a leucine could occur 85 times.
37
( Frequency of cleavages between two )( Frequency of cleavages between two specific residues
Total number of cleavages )( )Frequency of times two specific residues
appear next to each other in the sequences
=Normalized value ( )appear next to each other in the sequences
Total number of residues
( 9 )
Figure 2.3. Equation used for data normalization and example calculation from Keil(1993) Th li ti ti t k i t t th b d f i id
( 91344 )
( )4211240
=Normalized value = 1.8
(1993). The normalization equation takes into account the abundance of amino acids. The example calculation is for the probability of cleavage between a phenylalanine and a glycine. Cleavage between these two specific residues was observed nine times in the literature, where there was a total of 1344 cleavages. These two residues were found next to one another in the protein sequences 42 times. This means that there was possibility of cleavage between these two residues 42 times out of a possible 11240 residues. Therefore the probability of a cleavage occurring between a phenylalanine and
l i i 1 8a glycine is 1.8.
38
Table 2.5. Norm
alized cleavage data P
1 '
A
C
D
E F
G
H
I K
L
M
N
P Q
R
S
T V
W
Y A
0.5
1.2 0.3
0.93.5
0.00.5
2.20.6
1.60.8
0.3 0.2
0.20.5
0.50.4
1.70.0
4.2C
1.0
8.4 0.6
1.20.0
0.00.0
1.20.0
1.22.1
0.0 2.1
0.00.0
0.71.0
2.80.0
2.1D
1.1
1.0 0.5
0.71.9
0.10.0
2.50.0
0.80.5
1.2 1.2
0.90.5
0.20.9
1.72.8
2.4E
1.4 1.4
1.7 0.5
1.90.1
0.81.7
0.62.1
2.11.5
0.9 1.0
1.80.3
0.32.7
2.53.6
F 4.2
2.8 2.8
3.63.8
1.82.3
3.82.2
3.54.2
2.0 1.0
2.33.5
2.91.8
5.58.4
5.6G
0.6
1.5 0.0
0.41.0
0.40.6
0.70.1
0.70.6
0.2 0.0
0.20.4
0.50.3
0.41.5
1.0H
0.7
0.0 0.8
0.00.0
0.00.0
0.00.0
0.30.0
0.0 0.0
0.00.0
0.00.0
0.00.0
0.0I
0.9 0.0
0.0 0.3
1.70.0
0.00.5
0.20.7
0.71.0
0.0 0.3
0.50.6
0.60.6
0.00.4
K
0.3 0.0
0.3 0.6
0.00.0
0.00.2
0.20.1
0.00.5
0.0 0.0
0.00.2
0.70.1
0.00.3
P1
L 3.5
4.7 3.6
2.74.2
1.83.9
4.21.6
3.55.6
2.5 1.7
2.22.4
2.22.6
4.17.5
5.9M
2.3
4.2 1.3
3.13.6
0.71.4
2.82.7
1.24.8
2.8 0.0
0.00.0
2.30.6
2.88.4
3.6N
0.0
1.2 0.9
0.22.5
0.60.0
1.50.3
0.31.0
0.6 0.4
0.00.4
0.31.0
1.71.4
2.4P
0.0 0.0
0.2 0.0
0.00.0
0.00.4
0.00.0
0.00.5
0.0 0.0
0.00.0
0.30.3
0.00.0
Q
0.9 0.0
1.1 0.5
2.30.0
0.01.5
0.30.7
0.00.6
0.0 0.4
0.40.0
0.41.5
2.84.6
R
0.0 0.0
0.2 0.4
0.30.0
0.00.2
0.30.6
0.00.0
0.2 0.4
0.30.2
0.50.4
2.10.0
S 0.8
0.0 0.4
0.51.1
0.60.3
1.00.4
0.81.4
0.0 0.4
0.00.2
0.00.0
1.22.8
2.9T
1.0 0.0
1.2 0.2
1.70.8
0.01.5
0.00.9
0.50.4
0.0 0.4
1.20.0
0.00.4
0.92.6
V 0.7
0.0 0.9
0.10.5
0.20.0
0.50.4
0.40.5
0.0 0.0
0.40.2
0.50.7
0.41.4
2.5W
0.0
0.0 1.4
0.05.0
0.02.8
1.90.0
2.50.0
0.0 0.0
1.70.0
0.00.0
0.05.0
2.4Y
1.4 1.7
0.0 0.5
4.80.7
0.02.1
0.41.2
1.91.4
0.0 1.0
0.40.9
0.73.5
5.63.7
Residues in the P
1 positions are in the left hand column and residues in the P
1 ’ positions are along the top row.
39
Fi
P1
igure 2.4. Cleavage d
Figure 2.4 Cleavage
residues are on the ybetw
een phenylalan data map
e data map illustratin
y-axis while the P
1 ’ rnine and glycine
is 1.
P1 ’
ng the probability of cresidues are on the x-8 w
hile the probabili cleavage between tw
o-axis. For exam
ple, tity of cleavage betw
e o specific residues. Tthe probability of cleaeen leucine and trypto The P
1avage ophan is 7.5
40
The map shows that pepsin prefers to cleave most often after leucine,
phenylalanine, methionine and tyrosine. It prefers to cleave most often before
tryptophan, tyrosine, phenylalanine and valine. One of the more telling aspects of the
graph is where pepsin rarely cleaves. It is very obvious that pepsin will rarely cleave
after proline, histidine or lysine. Also, pepsin will rarely cleave before glycine, proline or
lysine.
2.6. Revised Cleavage Data Map
A second cleavage data map (Figure 2.5) was then produced illustrating the
probability of cleavage between two specific residues as a percentage. The percentages,
shown in Table 2.6, were calculated by dividing the number of times a cleavage occurred
between two specific amino acids by the total number of times those residues occurred
next to each other in the protein sequences. For example, cleavage between leucine and
tryptophan occurred nine out of a possible ten times, or 90% of the time while cleavage
between phenylalanine and glycine only occurred nine out of a possible 42 times, or 21%
of the time.
In the second cleavage data map presented the x-axis and y-axis are simply
ordered alphabetically. This illustration of the data makes it easier to spot trends in
pepsin specificity. While both of the cleavage data maps utilize color only in the second
version do those colors actually mean something. In the first version the colors were
arbitrarily assigned to a P1 residues while in the second version the colors represent the
percentage of time a cleavage occurred between two specific residues.
41
Figure 2.5. Cleavage data map with probability defined as a percentage
YWVTSR
P1
QPNMLKKIHGFED
Scale
A C D E F G H I K L M N P Q R S T V W Y
DC
A
P1’
100 90 80 70 60 50 40 30 20 10
Figure 2.5 Cleavage data map representing the probability of cleavage between two specific residues as a percentage. This map is read the same as in Figure 2.4 with the residue occurring before the cleavage site on the y-axis and the residue occurring following the cleavage site on the x-axis. Each colored square represents a percentagefollowing the cleavage site on the x axis. Each colored square represents a percentage range. For example, the brown square represents the range 21-30% and the orange square represents the range 81-90%. Cleavage between phenylalanine and glycineoccurred 21% of the time while cleavage between leucine and tryptophan occurred 90% of the time.
42
Table 2.6. Cleavage data w
ith probability defined as a percentage P
1 '
A
C
D
E F
G
H
I K
L
M
N
P Q
R
S
T V
W
Y A
6
14 4
11 42
0 6
26 7
19 9
3 3
3 6
6 4
20 0
50 C
13
100 8
14 0
0 0
14 0
14 25
0 25
0 0
8 13
33 0
25 D
13
13 6
8 22
2 0
30 0
9 6
14 15
11 6
3 11
20 33
29 E
17 17
20 6
23 2
10 21
8 25
25 18
11 12
21 3
4 32
30 43
F 50
33 33
43 45
21 27
46 26
42 50
24 12
28 42
34 22
65 100
67 G
7
18 0
5 11
5 7
8 2
8 7
3 0
3 5
6 3
5 18
12 H
8
0 10
0 0
0 0
0 0
3 0
0 0
0 0
0 0
0 0
0 I
11 0
0 4
21 0
0 6
3 8
8 13
0 4
6 7
7 7
0 5
K
4 0
4 7
0 0
0 3
3 1
0 6
0 0
0 3
9 2
0 4
P1
L 42
56 43
32 50
21 46
50 19
42 67
30 21
26 28
27 31
49 90
71 M
28
50 16
37 43
9 17
33 32
14 57
33 0
0 0
28 7
33 100
43 N
0
14 11
3 30
8 0
19 3
4 13
7 5
0 5
3 13
21 17
29 P
0 0
3 0
0 0
0 4
0 0
0 6
0 0
0 0
4 3
0 0
Q
11 0
13 6
27 0
0 18
3 9
0 7
0 5
5 0
5 18
33 56
R
0 0
3 4
4 0
0 3
3 7
0 0
3 4
3 3
6 5
25 0
S 10
0 5
6 13
7 4
11 5
9 17
0 4
0 3
0 0
14 33
35 T
12 0
15 2
20 10
0 18
0 10
6 5
0 5
14 0
0 5
11 32
V 8
0 11
2 6
2 0
5 5
4 6
0 0
5 3
6 8
5 17
29 W
0
0 17
0 60
0 33
22 0
30 0
0 0
20 0
0 0
0 60
29 Y
17 20
0 6
57 9
0 25
5 14
22 17
0 12
5 11
9 42
67 44
Residues in the P
1 positions are in the left hand column and residues in the P
1 ’ positions are along the top row. The values listed here
represent the percentage of time a cleavage w
as observed between tw
o specific residues.
43
2.7. Summary of Literature Search
The extensive search of contemporary scientific literature involving pepsin
digestions performed at pH 2.5 to 2.7 showed that pepsin does show some preferences in
where it cleaves. The peptic digest maps found in the literature were analyzed in order to
create a database of cleavages. The cleavages between the residues in the P1 and P1’
positions were tallied in order to create a matrix of data. These data were then
normalized in order to take into account the abundance of amino acids in the protein
sequences. The normalized data were then made into a cleavage map in order to easily
see trends in pepsin specificity. The literature shows that pepsin prefers to cleave after
bulky hydrophobic residues such as leucine and phenylalanine. It also shows that pepsin
will hardly ever cleave after proline or histidine.
The data gathered from the literature search only consists of 1,344 cleavages.
While this is a good starting point, much more data is needed to more accurately
determine trends in the specificity of pepsin.
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Rist, W., T. J. Jorgensen, et al. (2003). "Mapping temperature-induced conformational
changes in the Escherichia coli heat shock transcription factor sigma 32 by amide
Lu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) A A A G A V V GLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) G L G G Y M L GLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) Y M L G S A M SLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) L G S A M S R PLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) G S A M S R P ILu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) F G S D Y E D RLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) E D R Y Y R E NLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) Y Y R E N M H RLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) R E N M H R V PLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) Y P N Q V Y Y RLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) P N Q V Y Y R PLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) N Q V Y Y R P MLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) P M D E Y S N QLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) D C V N I T I KLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) C V N I T I K QLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) V N I T I K Q HLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) K G E N F T E TLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) F T E T D V K MLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) T E T D V K M MLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) D V K M M E R VLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) K M M E R V V ELu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) V E Q M C I T QLu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) C I T Q Y E R ELu, X., P. L. Wintrode, et al. (2007) Major prion protein (human) E S Q A Y Y Q RMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) M V L S E G EMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) S E E E W Q L VMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) E G G W Q L V LMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) Q L V L H V W AMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) V L H V W A K VMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) Q D I L I R L FMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) L I R L F K S HMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) E A E M K A S EMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) V T V L T A L GMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) L T A L G A I LMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) I P I K Y L E FMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) Y L E F I S E AMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) F I S E A I I HMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) I S E A I I H VMan, P., C. Montagner, et al. (2007) Myoglobin (sperm whale) A L E L F R K DBrier, S., E. Carletti, et al. (2006) CENP-E (human) M A E E GBrier, S., E. Carletti, et al. (2006) CENP-E (human) G A V A V C V RBrier, S., E. Carletti, et al. (2006) CENP-E (human) V A V C V R V RBrier, S., E. Carletti, et al. (2006) CENP-E (human) P L N S R E E SBrier, S., E. Carletti, et al. (2006) CENP-E (human) R P L N S R E EBrier, S., E. Carletti, et al. (2006) CENP-E (human) N S R E E S L GBrier, S., E. Carletti, et al. (2006) CENP-E (human) E T A Q V Y W KBrier, S., E. Carletti, et al. (2006) CENP-E (human) T A Q V Y W K TBrier, S., E. Carletti, et al. (2006) CENP-E (human) K T D N N V I YBrier, S., E. Carletti, et al. (2006) CENP-E (human) T D N N V I Y QBrier, S., E. Carletti, et al. (2006) CENP-E (human) N V I Y Q V D G
51
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Brier, S., E. Carletti, et al. (2006) CENP-E (human) I Y Q V D G S KBrier, S., E. Carletti, et al. (2006) CENP-E (human) K S F N F D R VBrier, S., E. Carletti, et al. (2006) CENP-E (human) S F N F D R V FBrier, S., E. Carletti, et al. (2006) CENP-E (human) T T K N V Y E EBrier, S., E. Carletti, et al. (2006) CENP-E (human) K N V Y E E I ABrier, S., E. Carletti, et al. (2006) CENP-E (human) N V Y E E I A ABrier, S., E. Carletti, et al. (2006) CENP-E (human) V Y E E I A A PBrier, S., E. Carletti, et al. (2006) CENP-E (human) E I A A P I I DBrier, S., E. Carletti, et al. (2006) CENP-E (human) P I I D S A I QBrier, S., E. Carletti, et al. (2006) CENP-E (human) I I D S A I Q GBrier, S., E. Carletti, et al. (2006) CENP-E (human) I D S A I Q G YBrier, S., E. Carletti, et al. (2006) CENP-E (human) G T I F A Y G QBrier, S., E. Carletti, et al. (2006) CENP-E (human) T I F A Y G Q TBrier, S., E. Carletti, et al. (2006) CENP-E (human) S G K T Y T M MBrier, S., E. Carletti, et al. (2006) CENP-E (human) Y T M M G S E DBrier, S., E. Carletti, et al. (2006) CENP-E (human) E D H L G V I PBrier, S., E. Carletti, et al. (2006) CENP-E (human) H D I F Q K I KBrier, S., E. Carletti, et al. (2006) CENP-E (human) R E F L L R V SBrier, S., E. Carletti, et al. (2006) CENP-E (human) E F L L R V S YBrier, S., E. Carletti, et al. (2006) CENP-E (human) R V S Y M E I YBrier, S., E. Carletti, et al. (2006) CENP-E (human) V S Y M E I Y NBrier, S., E. Carletti, et al. (2006) CENP-E (human) I T D L L C G TBrier, S., E. Carletti, et al. (2006) CENP-E (human) T D L L C G T QBrier, S., E. Carletti, et al. (2006) CENP-E (human) I I R E D V N RBrier, S., E. Carletti, et al. (2006) CENP-E (human) R N V Y V A D LBrier, S., E. Carletti, et al. (2006) CENP-E (human) Y V A D L T E EBrier, S., E. Carletti, et al. (2006) CENP-E (human) V A D L T E E VBrier, S., E. Carletti, et al. (2006) CENP-E (human) L T E E V V Y TBrier, S., E. Carletti, et al. (2006) CENP-E (human) Y T S E M A L KBrier, S., E. Carletti, et al. (2006) CENP-E (human) T S E M A L K WBrier, S., E. Carletti, et al. (2006) CENP-E (human) E T K M N Q R SBrier, S., E. Carletti, et al. (2006) CENP-E (human) H T I F R M I LBrier, S., E. Carletti, et al. (2006) CENP-E (human) R M I L E S R EBrier, S., E. Carletti, et al. (2006) CENP-E (human) P S N C E G S VBrier, S., E. Carletti, et al. (2006) CENP-E (human) V S H L N L V DBrier, S., E. Carletti, et al. (2006) CENP-E (human) S H L N L V D LBrier, S., E. Carletti, et al. (2006) CENP-E (human) H L N L V D L ABrier, S., E. Carletti, et al. (2006) CENP-E (human) A G S E R A A QBrier, S., E. Carletti, et al. (2006) CENP-E (human) A A Q T G A A GBrier, S., E. Carletti, et al. (2006) CENP-E (human) G A A G V R L KBrier, S., E. Carletti, et al. (2006) CENP-E (human) V R L K E G C NBrier, S., E. Carletti, et al. (2006) CENP-E (human) E G C N I N R SBrier, S., E. Carletti, et al. (2006) CENP-E (human) G C N I N R S LBrier, S., E. Carletti, et al. (2006) CENP-E (human) N R S L F I L GBrier, S., E. Carletti, et al. (2006) CENP-E (human) R S L F I L G QBrier, S., E. Carletti, et al. (2006) CENP-E (human) V G G F I N Y RBrier, S., E. Carletti, et al. (2006) CENP-E (human) G G F I N Y R DBrier, S., E. Carletti, et al. (2006) CENP-E (human) F I N Y R D S KBrier, S., E. Carletti, et al. (2006) CENP-E (human) D S K L T R I LBrier, S., E. Carletti, et al. (2006) CENP-E (human) N A K T R I I CBrier, S., E. Carletti, et al. (2006) CENP-E (human) R I I C T I T P
52
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Brier, S., E. Carletti, et al. (2006) CENP-E (human) I I C T I T P VBrier, S., E. Carletti, et al. (2006) CENP-E (human) I T P V S F D EBrier, S., E. Carletti, et al. (2006) CENP-E (human) P V S F D E T LBrier, S., E. Carletti, et al. (2006) CENP-E (human) T L T A L Q F ABrier, S., E. Carletti, et al. (2006) CENP-E (human) L T A L Q F A SBrier, S., E. Carletti, et al. (2006) CENP-E (human) T A L Q F A S TBrier, S., E. Carletti, et al. (2006) CENP-E (human) A L Q F A S T ABrier, S., E. Carletti, et al. (2006) CENP-E (human) F A S T A K Y MBrier, S., E. Carletti, et al. (2006) CENP-E (human) Y V N E V S T DBrier, S., E. Carletti, et al. (2006) CENP-E (human) V N E V S T D LCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein H V A F G S E DCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein F G S E D I E NCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein G S E D I E N TCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein E N T L A K M DCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein D G Q L D G L ACheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein L D G L A F G ACheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein A I Q L D G D GCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein N I L Q Y N A ACheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein I L Q Y N A A ECheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein I G K N F F K DCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein G K N F F K D VCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein S P E F Y G K FCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein S G N L N T M FCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein N L N T M F E YCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein L N T M F E Y TCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein E Y T F D Y Q MCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein Y T F D Y Q M TCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein S G D S Y W V FCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein G D S Y W V F VCheng, G., M. A. Cusanovich, et al. (2006) Photoactive yellow protein Y W V F V K R VCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus G D A A K G E KCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus H S I I A P D GCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus D G T E I V K GCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus G P N L Y G V VCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus Y P E F K Y K DCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus I V A L G A S GCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus S I V A L G A SCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus G A S G F A W TCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus A S G F A W T ECheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus W T E E D I A TCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus T E E D I A T YCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus D I A T Y V K DCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus P G A F L K E KCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus G A F L K E K LCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus T G M A F K L ACheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus E D V A A Y L ACheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus D V A A Y L A SCheng, G., V. H. Wysocki, et al. (2006) cytochrome c 2-rhodobacter capsulatus Y L A S V V KHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef G W S A I R E RHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef D G V G A V S RHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef N A D C A W L E
53
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Hochrein, J. M., T. E. Wales, et al. (2006) HIV Nef A Q E E E E V GHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef R P M T Y K A AHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef K A A L D I S HHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef I S H F L K E KHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef K G G L E G L IHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef I L D L W I Y HHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef L D L W I Y H THochrein, J. M., T. E. Wales, et al. (2006) HIV Nef Q G Y F P D W QHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef D W Q N Y T P GHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef L T F G W C F KHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef G W C F K L V PHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef K V E E A N E GHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef P M S L H G M EHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef K E V L V W R FHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef D S K L A F H HHochrein, J. M., T. E. Wales, et al. (2006) HIV Nef H P E Y Y K D CHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef H G G A I S M RHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef G D L R Q R L LHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef Y G R L L G E VHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef L S S L S C E GHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef N Q G Q Y M N THochrein, J. M., T. E. Wales, et al. (2006) SIV Nef P A E E R E K LHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef N M D D I D E EHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef D D D L V G V SHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef R T M S Y K L AHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef S Y K L A I D MHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef M S H F I K E KHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef E G I Y Y S A RHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef R I L D I Y L EHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef D I Y L E K E EHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef D W Q D Y T S GHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef F G W L W K L VHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef E A Q E D E E HHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef Q T S Q W D D PHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef Y E A Y V R Y PHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef K S G L S E E EHochrein, J. M., T. E. Wales, et al. (2006) SIV Nef L L N M A D K KShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis R A D I L Y N IShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis A D I L Y N I RShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis R P V A V S V SShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis P V A V S V S LShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis D V V F W Q Q TShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis S D R T L A W NShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis D L A A Y N A IShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis T P Q L A R V VShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis G E V L Y M P SShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis D D S E Y F S QShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis D S E Y F S Q YShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis Y S R F E I L DShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis F E I L D V T QShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis E A Y E D V E V
54
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Shi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis E D V E V S L NShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis E V S L N F R KShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis V S L N F R K KShi, J., J. R. Koeppe, et al. (2006) AChBP from L. stagnalis S L N F R K K GTsutsui, Y., L. Liu, et al. (2006) α1AT (human) F N K I TTsutsui, Y., L. Liu, et al. (2006) α1AT (human) N L A E F A F STsutsui, Y., L. Liu, et al. (2006) α1AT (human) A F S L Y R Q LTsutsui, Y., L. Liu, et al. (2006) α1AT (human) S T N I F F S PTsutsui, Y., L. Liu, et al. (2006) α1AT (human) I A T A F A M LTsutsui, Y., L. Liu, et al. (2006) α1AT (human) A F A M L S L GTsutsui, Y., L. Liu, et al. (2006) α1AT (human) G T K A D T H DTsutsui, Y., L. Liu, et al. (2006) α1AT (human) D E I L E G L NTsutsui, Y., L. Liu, et al. (2006) α1AT (human) N F N L T E I PTsutsui, Y., L. Liu, et al. (2006) α1AT (human) Q E L L H T L NTsutsui, Y., L. Liu, et al. (2006) α1AT (human) N G L F L S E GTsutsui, Y., L. Liu, et al. (2006) α1AT (human) L K L V D K F LTsutsui, Y., L. Liu, et al. (2006) α1AT (human) H S E A F T V NTsutsui, Y., L. Liu, et al. (2006) α1AT (human) Q I N D Y V E KTsutsui, Y., L. Liu, et al. (2006) α1AT (human) I V D L V K E LTsutsui, Y., L. Liu, et al. (2006) α1AT (human) D T V F A L V NTsutsui, Y., L. Liu, et al. (2006) α1AT (human) Y I F F K G K NTsutsui, Y., L. Liu, et al. (2006) α1AT (human) E E D F H V D QTsutsui, Y., L. Liu, et al. (2006) α1AT (human) Q V T T V K V PTsutsui, Y., L. Liu, et al. (2006) α1AT (human) L G M F N I Q HTsutsui, Y., L. Liu, et al. (2006) α1AT (human) K L S S W V L LTsutsui, Y., L. Liu, et al. (2006) α1AT (human) S W V L L M K YTsutsui, Y., L. Liu, et al. (2006) α1AT (human) A T A I F F L PTsutsui, Y., L. Liu, et al. (2006) α1AT (human) I T K F L E N ETsutsui, Y., L. Liu, et al. (2006) α1AT (human) T Y D L K S V LTsutsui, Y., L. Liu, et al. (2006) α1AT (human) K S V L G Q L GTsutsui, Y., L. Liu, et al. (2006) α1AT (human) N G A D L S G VTsutsui, Y., L. Liu, et al. (2006) α1AT (human) V T E E A P L KTsutsui, Y., L. Liu, et al. (2006) α1AT (human) L K L S K A V HTsutsui, Y., L. Liu, et al. (2006) α1AT (human) A V L T I D E KTsutsui, Y., L. Liu, et al. (2006) α1AT (human) A G A M F L E ATsutsui, Y., L. Liu, et al. (2006) α1AT (human) P F V F L M I DTsutsui, Y., L. Liu, et al. (2006) α1AT (human) S P L F M G K VWales, T. E. and J. R. Engen (2006) Lyn SH3 D I V V A L Y PWales, T. E. and J. R. Engen (2006) Lyn SH3 F K K G E K M KWales, T. E. and J. R. Engen (2006) Lyn SH3 H G E W W K A KWales, T. E. and J. R. Engen (2006) Lyn SH3 E W W K A K S LWales, T. E. and J. R. Engen (2006) Lyn SH3 K S L L T K E EWales, T. E. and J. R. Engen (2006) Lyn SH3 K E G F I P S NWales, T. E. and J. R. Engen (2006) Lyn SH3 P S N Y V A K LWales, T. E. and J. R. Engen (2006) Lyn SH3 A K L N T L E TWales, T. E. and J. R. Engen (2006) α-spectrin SH3 A L Y D Y Q E KWales, T. E. and J. R. Engen (2006) α-spectrin SH3 K S P R E V T MWales, T. E. and J. R. Engen (2006) α-spectrin SH3 P R E V T M K KWales, T. E. and J. R. Engen (2006) α-spectrin SH3 G D I L T L L NWales, T. E. and J. R. Engen (2006) α-spectrin SH3 D I L T L L N SWales, T. E. and J. R. Engen (2006) α-spectrin SH3 L N S T N K D W
55
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Wales, T. E. and J. R. Engen (2006) α-spectrin SH3 T N K D W W K VWales, T. E. and J. R. Engen (2006) α-spectrin SH3 D R Q G F V P AWales, T. E. and J. R. Engen (2006) α-spectrin SH3 A A Y V K K L DWeis, D. D., P. Kjellen, et al. (2006) Lck SH3 N L V I A LWeis, D. D., P. Kjellen, et al. (2006) Lck SH3 V I A L H S Y EWeis, D. D., P. Kjellen, et al. (2006) Lck SH3 K G E Q L R I LWeis, D. D., P. Kjellen, et al. (2006) Lck SH3 G E Q L R I L EWeis, D. D., P. Kjellen, et al. (2006) Lck SH3 S G E W W K A QWeis, D. D., P. Kjellen, et al. (2006) Lck SH3 G F I P F N F VWeis, D. D., P. Kjellen, et al. (2006) Lck SH3 P F N F V A K AYao, Z. P., M. Zhou, et al. (2006) Cks1 D D E E F E Y RYao, Z. P., M. Zhou, et al. (2006) Cks1 K T H L M S E SYao, Z. P., M. Zhou, et al. (2006) Cks1 S E S E W R N LYao, Z. P., M. Zhou, et al. (2006) Cks1 Q S Q G W V H YYao, Z. P., M. Zhou, et al. (2006) Cks1 W V H Y M I H EYao, Z. P., M. Zhou, et al. (2006) Cks1 H I L L F R R PYao, Z. P., M. Zhou, et al. (2006) Skp2 G S P E F MYao, Z. P., M. Zhou, et al. (2006) Skp2 P K L N R E N FYao, Z. P., M. Zhou, et al. (2006) Skp2 P G V S W D S LYao, Z. P., M. Zhou, et al. (2006) Skp2 D E L L L G I FYao, Z. P., M. Zhou, et al. (2006) Skp2 C L P E L L K VYao, Z. P., M. Zhou, et al. (2006) Skp2 S D E S L W Q TYao, Z. P., M. Zhou, et al. (2006) Skp2 V T G R L L S QYao, Z. P., M. Zhou, et al. (2006) Skp2 V I A F R C P RYao, Z. P., M. Zhou, et al. (2006) Skp2 P L A E H F S PYao, Z. P., M. Zhou, et al. (2006) Skp2 H M D L S N S VYao, Z. P., M. Zhou, et al. (2006) Skp2 S V I E V S T LYao, Z. P., M. Zhou, et al. (2006) Skp2 H G I L S Q C SYao, Z. P., M. Zhou, et al. (2006) Skp2 N L S L E G L RYao, Z. P., M. Zhou, et al. (2006) Skp2 V N T L A K N SYao, Z. P., M. Zhou, et al. (2006) Skp2 N S N L V R L NYao, Z. P., M. Zhou, et al. (2006) Skp2 S G C S G F S EYao, Z. P., M. Zhou, et al. (2006) Skp2 F S E F A L Q TYao, Z. P., M. Zhou, et al. (2006) Skp2 Q T L L S S C SYao, Z. P., M. Zhou, et al. (2006) Skp2 S C S R L D E LYao, Z. P., M. Zhou, et al. (2006) Skp2 A H V S E T I TYao, Z. P., M. Zhou, et al. (2006) Skp2 I T Q L N L S GYao, Z. P., M. Zhou, et al. (2006) Skp2 K S D L S T L VYao, Z. P., M. Zhou, et al. (2006) Skp2 R R S P N L V HYao, Z. P., M. Zhou, et al. (2006) Skp2 L D L S D S V MYao, Z. P., M. Zhou, et al. (2006) Skp2 L K N D C F Q EYao, Z. P., M. Zhou, et al. (2006) Skp2 F Q E F F Q L NYao, Z. P., M. Zhou, et al. (2006) Skp2 E F F Q L N Y LYao, Z. P., M. Zhou, et al. (2006) Skp2 Q L N Y L Q H LYao, Z. P., M. Zhou, et al. (2006) Skp2 H L S L S R C YYao, Z. P., M. Zhou, et al. (2006) Skp2 L S R C Y D I IYao, Z. P., M. Zhou, et al. (2006) Skp2 P E T L L E L GYao, Z. P., M. Zhou, et al. (2006) Skp2 P T L K T L Q VYao, Z. P., M. Zhou, et al. (2006) Skp2 L Q V F G I V PYao, Z. P., M. Zhou, et al. (2006) Skp2 T L Q L L K E AYao, Z. P., M. Zhou, et al. (2006) Skp2 L P H L Q I N C
56
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Yao, Z. P., M. Zhou, et al. (2006) Skp2 K C R L T L Q KCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 F F F G N ICatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 I T R E E A E DCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 T R E E A E D YCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 G M S D G L Y LCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 S D G L Y L L RCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 Q S R N Y L G GCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 Y L G G F A L SCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 L G G F A L S VCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 H Y T I L N G TCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 N G T Y A I A GCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 P A D L C H Y HCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 E S D G L V C LCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 L V C L L K K PCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 F E D L K E N LCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 K E N L I R E YCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 E N L I R E Y VCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 I R E Y V K Q TCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 Q A L E Q A I LCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 A L E Q A I L SCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 Q A I L S Q K PCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 K P Q L E K L ICatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 L E K L I A T TCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 E K L I A T T ACatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 S R D E S E Q ICatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 D E S E Q I V LCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 S E Q I V L I GCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 Q I V L I G S KCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 N G K F L I R ACatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 N N G S Y A L CCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 S Y A L C L L HCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 F D T L W Q L VCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 E H Y S Y K S DCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 S D G L L R V LCatalina, M. I., M. J. Fischer, et al. (2005) SH2 domains of Syk tSH2 D G L L R V L TKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein I V T L A V D EKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein L A V D E I I EKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein A S M Q R S S NKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein I W M P V E Q EKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein P V E Q E S P TKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein W D L T D K A TKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein A T G L L E L NKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein D N D F F Q L RKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein D L R D E T A YKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein N N V E L K V AKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein N V E L K V A NKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein G S L V I T S PKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein D A W N F V A DKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein E I M F S R E LKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein G T S Y F F N PKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein D I F G R I P E
57
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Kang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein P E E A Y R D GKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein V A G F D D V LKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein V D N R F A T VKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein T V T L S A T TKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein K I S F A G V KKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein A K N V L A Q DKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein D A T F S V V RKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein P V A L D D V SKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein A D A M A V N IKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein T N V F W A D DKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein W A D D A I R IKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein H E L F A G M KKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein F A G M K T T SKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein N G I F A T Q GKang, S. and P. E. Prevelige, Jr. (2005) P22 capsid coat protein L S G L C R I ALee, T., A. N. Hoofnagle, et al. (2005) ERK2 A G P E M V R GLee, T., A. N. Hoofnagle, et al. (2005) ERK2 G M V C S A Y DLee, T., A. N. Hoofnagle, et al. (2005) ERK2 V C S A Y D N LLee, T., A. N. Hoofnagle, et al. (2005) ERK2 Y D N L N K V RLee, T., A. N. Hoofnagle, et al. (2005) ERK2 T L R E I K I LLee, T., A. N. Hoofnagle, et al. (2005) ERK2 I K I L L R F RLee, T., A. N. Hoofnagle, et al. (2005) ERK2 G I N D I I R ALee, T., A. N. Hoofnagle, et al. (2005) ERK2 K D V Y I V Q DLee, T., A. N. Hoofnagle, et al. (2005) ERK2 V Q D L M E T DLee, T., A. N. Hoofnagle, et al. (2005) ERK2 D L M E T D L YLee, T., A. N. Hoofnagle, et al. (2005) ERK2 E T D L Y K L LLee, T., A. N. Hoofnagle, et al. (2005) ERK2 H I C Y F L Y QLee, T., A. N. Hoofnagle, et al. (2005) ERK2 A N V L H R D LLee, T., A. N. Hoofnagle, et al. (2005) ERK2 P S N L L L N TLee, T., A. N. Hoofnagle, et al. (2005) ERK2 S N L L L N T TLee, T., A. N. Hoofnagle, et al. (2005) ERK2 T T C D L K I CLee, T., A. N. Hoofnagle, et al. (2005) ERK2 T C D L K I C DLee, T., A. N. Hoofnagle, et al. (2005) ERK2 D F G L A R V ALee, T., A. N. Hoofnagle, et al. (2005) ERK2 A T R W Y R A PLee, T., A. N. Hoofnagle, et al. (2005) ERK2 P E I N L N S KLee, T., A. N. Hoofnagle, et al. (2005) ERK2 E I N L N S K GLee, T., A. N. Hoofnagle, et al. (2005) ERK2 W S V G C I L ALee, T., A. N. Hoofnagle, et al. (2005) ERK2 L A E M L S N RLee, T., A. N. Hoofnagle, et al. (2005) ERK2 P S Q E D L N CLee, T., A. N. Hoofnagle, et al. (2005) ERK2 Q E D L N C I ILee, T., A. N. Hoofnagle, et al. (2005) ERK2 I I N L K A R NLee, T., A. N. Hoofnagle, et al. (2005) ERK2 R N Y L L S L PLee, T., A. N. Hoofnagle, et al. (2005) ERK2 A L D L L D K MLee, T., A. N. Hoofnagle, et al. (2005) ERK2 Y L E Q Y Y D PLee, T., A. N. Hoofnagle, et al. (2005) ERK2 D M E L D D L PLee, T., A. N. Hoofnagle, et al. (2005) ERK2 E L I F E E T ALee, T., A. N. Hoofnagle, et al. (2005) ERK2 I C D F G L A RBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) K N I Q V V V RBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) N I Q V V V R CBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) V R C R P F N LBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) P F N K A E R K
58
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Brier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) K A S A H S I VBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) S A H S I V E CBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) S I V E C D P VBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) V E C D P V R KBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) K E V S V R T GBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) V R T G G L A DBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) T G G L A D K SBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) S S R K T Y T FBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) T Y T F D M V FBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) Y T F D M V F GBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) F D M V F G A SBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) Q I D V Y R S VBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) S V V C P I L DBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) C P I L D E V IBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) P I L D E V I MBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) I L D E V I M GBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) D E V I M G Y NBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) M G Y N C T I FBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) N C T I F A Y GBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) C T I F A Y G QBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) Y G Q T G T G KBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) P N E E Y T W EBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) N E E Y T W E EBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) E E Y T W E E DBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) Y T W E E D P LBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) I I P R T L H QBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) P R T L H Q I FBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) T L H Q I F E KBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) H Q I F E K L TBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) Q I F E K L T DBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) K L T D N G T EBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) N G T E F S V KBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) G T E F S V K VBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) T E F S V K V SBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) K V S L L E I YBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) V S L L E I Y NBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) N E E L F D L LBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) N P S S D V S EBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) D V S E R L Q MBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) R L Q M F D D PBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) K R G V I I K GBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) G L E E I T V HBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) V H N K D E V YBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) D E V Y Q I L EBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) E V Y Q I L E KBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) Y Q I L E K G ABrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) Q I L E K G A ABrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) A A T L M N A YBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) A T L M N A Y SBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) L M N A Y S S RBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) S S R S H S V F
59
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Brier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) H S V F S V T IBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) T I H M K E T TBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) T I D G E E L VBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) D G E E L V K IBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) G E E L V K I GBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) E E L V K I G KBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) K L N L V D L ABrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) L N L V D L A GBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) N I G R S G A VBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) R E A G N I N QBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) N Q S L L T L GBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) Q S L L T L G RBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) L L T L G R V IBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) L T L G R V I TBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) R V I T A L V EBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) V I T A L V E RBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) I T A L V E R TBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) R E S K L T R IBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) E S K L T R I LBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) L Q D S L G G RBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) Q D S L G G R TBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) D S L G G R T RBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) G G R T R T S IBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) R T S I I A T IBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) T S I I A T I SBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) S I I A T I S PBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) I I A T I S P ABrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) I A T I S P A SBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) I S P A S L N LBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) S P A S L N L EBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) S L N L E E T LBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) L N L E E T L SBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) E E T L S T L EBrier, S., D. Lemaire, et al. (2004) Kinesin-like protein KIF11 (human) S T L E Y A G RCasbarra, A., L. Birolo, et al. (2004) Human α-LA L S Q L L K D ICasbarra, A., L. Birolo, et al. (2004) Human α-LA G I A L P E L ICasbarra, A., L. Birolo, et al. (2004) Human α-LA C T M F H T S GCasbarra, A., L. Birolo, et al. (2004) Human α-LA Y G L F Q I S NCasbarra, A., L. Birolo, et al. (2004) Human α-LA S N K L W C K SCasbarra, A., L. Birolo, et al. (2004) Human α-LA K I L D I K G ICasbarra, A., L. Birolo, et al. (2004) Human α-LA G I D Y W L A HCasbarra, A., L. Birolo, et al. (2004) Human α-LA L E Q W L C E KCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin R P L F E K K SCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin T E R E L L E SCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin L E S Y I D G RCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin E G S D A E I GCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin Q V M L F R K SCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin Q E L L C G A SCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin N D L L V R I GCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin D L L V R I G KCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin E R N I E K I S
60
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Croy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin E K I S M L E KCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin I S M L E K I YCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin S M L E K I Y ICroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin Y N W R E N L DCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin R E N L D R D ICroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin D I A L M K L KCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin F S D Y I H P VCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin A A S L L Q A GCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin A S L L Q A G YCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin G N L K E T W TCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin G G P F V M K SCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin P F V M K S P FCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin F N N R W Y Q MCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin K Y G F Y T H VCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin T H V F R K K KCroy, C. H., J. R. Koeppe, et al. (2004) human α-thrombin I D Q F G ECroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin Q P F F N E K TCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin V M L F R K S PCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin Q E L L C G A SCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin A S L I S D R WCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin W V L T A A H CCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin D D L L V R I GCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin K E N L D R D ICroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin D I A L L K L KCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin R P I E L S D YCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin L S D Y I H P VCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin H A G F K G R VCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin T T S V A E V QCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin T S V A E V Q PCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin K A S T R I R ICroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin T D N M F C A GCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin D N M F C A G YCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin G G P F V M K SCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin P F V M K S P YCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin K S P Y N N R WCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin Y N N R W Y Q MCroy, C. H., J. R. Koeppe, et al. (2004) bovine α-thrombin G I V S W G E GCroy, C. H., S. Bergqvist, et al. (2004) IκBα D S F L H L A ICroy, C. H., S. Bergqvist, et al. (2004) IκBα F L H L A I I HCroy, C. H., S. Bergqvist, et al. (2004) IκBα A L T M E V I RCroy, C. H., S. Bergqvist, et al. (2004) IκBα L T M E V I R QCroy, C. H., S. Bergqvist, et al. (2004) IκBα M E V I R Q V KCroy, C. H., S. Bergqvist, et al. (2004) IκBα K G D L A F L NCroy, C. H., S. Bergqvist, et al. (2004) IκBα D L A F L N F QCroy, C. H., S. Bergqvist, et al. (2004) IκBα L A F L N F Q NCroy, C. H., S. Bergqvist, et al. (2004) IκBα F L N F Q N N LCroy, C. H., S. Bergqvist, et al. (2004) IκBα P L H L A V I TCroy, C. H., S. Bergqvist, et al. (2004) IκBα E I A E A L L GCroy, C. H., S. Bergqvist, et al. (2004) IκBα A G C D P E L RCroy, C. H., S. Bergqvist, et al. (2004) IκBα D P E L R D F RCroy, C. H., S. Bergqvist, et al. (2004) IκBα E L R D F R G N
61
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Croy, C. H., S. Bergqvist, et al. (2004) IκBα G N T R L H L ACroy, C. H., S. Bergqvist, et al. (2004) IκBα T P K H L A C ECroy, C. H., S. Bergqvist, et al. (2004) IκBα P L H L A C E QCroy, C. H., S. Bergqvist, et al. (2004) IκBα E Q G C L A S VCroy, C. H., S. Bergqvist, et al. (2004) IκBα L A S V G V L TCroy, C. H., S. Bergqvist, et al. (2004) IκBα L H S I L K A TCroy, C. H., S. Bergqvist, et al. (2004) IκBα G H T L L H L ACroy, C. H., S. Bergqvist, et al. (2004) IκBα H T L L H L A SCroy, C. H., S. Bergqvist, et al. (2004) IκBα G I V E L L V SCroy, C. H., S. Bergqvist, et al. (2004) IκBα I V E L L V S KCroy, C. H., S. Bergqvist, et al. (2004) IκBα G R T A L H L ACroy, C. H., S. Bergqvist, et al. (2004) IκBα A L H L A V D KCroy, C. H., S. Bergqvist, et al. (2004) IκBα R P S T R I Q QCroy, C. H., S. Bergqvist, et al. (2004) IκBα Q L G Q L T L ECroy, C. H., S. Bergqvist, et al. (2004) IκBα E S E F T E F TCroy, C. H., S. Bergqvist, et al. (2004) IκBα Y D D C V F G GLi, X., Y. T. Chou, et al. (2004) LR3IGF-I M P S L F V N GLi, X., Y. T. Chou, et al. (2004) LR3IGF-I P S L F V N G PLi, X., Y. T. Chou, et al. (2004) LR3IGF-I C G A E L V D ALi, X., Y. T. Chou, et al. (2004) LR3IGF-I G A E L V D A LLi, X., Y. T. Chou, et al. (2004) LR3IGF-I L V D A L Q F VLi, X., Y. T. Chou, et al. (2004) LR3IGF-I V D A L Q F V CLi, X., Y. T. Chou, et al. (2004) LR3IGF-I D A L Q E V C GLi, X., Y. T. Chou, et al. (2004) LR3IGF-I D R G F Y F N KLi, X., Y. T. Chou, et al. (2004) LR3IGF-I R G F Y F N K PLi, X., Y. T. Chou, et al. (2004) LR3IGF-I F Y F N K P T GLi, X., Y. T. Chou, et al. (2004) LR3IGF-I Q T G I V D E CLi, X., Y. T. Chou, et al. (2004) LR3IGF-I V D E C C F R SLi, X., Y. T. Chou, et al. (2004) LR3IGF-I R L E M Y C A PMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) Y P D L S K H NMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) T P D L Y K K LMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) E T P S G F T LMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) P S G F T L D DMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) F T L D D V I QMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) T L D D V I Q TMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) F I M T V G C VMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) M T V G C V A GMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) D E E S Y T V FMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) Y T V F K D L FMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) D L F D P I I QMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) H E N L K G G DMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) H Y V L S S R VMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) K L S V E A L NMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) V E A L N S L TMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) Q Q Q L I D D HMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) D D H F L F D KMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) D H F L F D K PMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) S P L L L A S GMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) P L L L A S G MMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) N K S F L V W VMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) K S F L V W V N
62
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Mazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) E D H L R V I SMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) V I S M E K Q GMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) K E V F R R F CMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) F C V G L Q K IMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) K I E E I F K KMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) H P F M W N E HMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) H L G Y V L T CMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) G Y V L T C P SMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) V L T C P S N LMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) P S N L G T G LMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) H P K F E E I LMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) E E I L T R L RMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) L T R L R L Q KMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) A A V G S V F DMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) G S V F D I S NMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) S E V E Q V Q LMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) E V E Q V Q L VMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) Q V Q L V V D GMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) G V K L M V E MMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) L M V E M E K KMazon, H., O. Marcillat, et al. (2004) Creatine kinase M-type (rabbit) M V E M E K K LWu, Z., A. Hasan, et al. (2004) human rCRALBP D K H M S E G VWu, Z., A. Hasan, et al. (2004) human rCRALBP Y V N F R L Q YWu, Z., A. Hasan, et al. (2004) human rCRALBP Q A E N T A FYan, X., D. Broderick, et al. (2004) human RXRα LBD M P V E R I L EYan, X., D. Broderick, et al. (2004) human RXRα LBD P V E R I L E AYan, X., D. Broderick, et al. (2004) human RXRα LBD E R I L E A E LYan, X., D. Broderick, et al. (2004) human RXRα LBD I L E A E L A VYan, X., D. Broderick, et al. (2004) human RXRα LBD L E A E L A V EYan, X., D. Broderick, et al. (2004) human RXRα LBD E A E L A V E PYan, X., D. Broderick, et al. (2004) human RXRα LBD K T E T Y V E AYan, X., D. Broderick, et al. (2004) human RXRα LBD V E A N M G L NYan, X., D. Broderick, et al. (2004) human RXRα LBD N M G L N P S SYan, X., D. Broderick, et al. (2004) human RXRα LBD N I C Q A A D KYan, X., D. Broderick, et al. (2004) human RXRα LBD Q L F T L V F WYan, X., D. Broderick, et al. (2004) human RXRα LBD L F T L V E W AYan, X., D. Broderick, et al. (2004) human RXRα LBD T L V E W A K RYan, X., D. Broderick, et al. (2004) human RXRα LBD I P H F S E L PYan, X., D. Broderick, et al. (2004) human RXRα LBD P H F S E L P LYan, X., D. Broderick, et al. (2004) human RXRα LBD F S E L P L D DYan, X., D. Broderick, et al. (2004) human RXRα LBD P L D D Q V I LYan, X., D. Broderick, et al. (2004) human RXRα LBD L D D Q V I L LYan, X., D. Broderick, et al. (2004) human RXRα LBD Q V I L L R A GYan, X., D. Broderick, et al. (2004) human RXRα LBD W N E L L I A SYan, X., D. Broderick, et al. (2004) human RXRα LBD N E L L I A S FYan, X., D. Broderick, et al. (2004) human RXRα LBD L I A S F S H RYan, X., D. Broderick, et al. (2004) human RXRα LBD I A S F S H R SYan, X., D. Broderick, et al. (2004) human RXRα LBD R S I A V K D GYan, X., D. Broderick, et al. (2004) human RXRα LBD D G I L L A T GYan, X., D. Broderick, et al. (2004) human RXRα LBD G I L L A T G LYan, X., D. Broderick, et al. (2004) human RXRα LBD A T G L H V H R
63
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Yan, X., D. Broderick, et al. (2004) human RXRα LBD A G V G A I F DYan, X., D. Broderick, et al. (2004) human RXRα LBD G A I F D R V LYan, X., D. Broderick, et al. (2004) human RXRα LBD A I F D R V L TYan, X., D. Broderick, et al. (2004) human RXRα LBD V L T E L V S KYan, X., D. Broderick, et al. (2004) human RXRα LBD L T E L V S K MYan, X., D. Broderick, et al. (2004) human RXRα LBD D M Q M D K T EYan, X., D. Broderick, et al. (2004) human RXRα LBD K T E L G C L RYan, X., D. Broderick, et al. (2004) human RXRα LBD L G C L R A I VYan, X., D. Broderick, et al. (2004) human RXRα LBD C L R A I V L FYan, X., D. Broderick, et al. (2004) human RXRα LBD A I V L F N P DYan, X., D. Broderick, et al. (2004) human RXRα LBD N P A E V E A LYan, X., D. Broderick, et al. (2004) human RXRα LBD A E V E A L R EYan, X., D. Broderick, et al. (2004) human RXRα LBD E V E A L R E KYan, X., D. Broderick, et al. (2004) human RXRα LBD A L R E K V Y AYan, X., D. Broderick, et al. (2004) human RXRα LBD Y A S L E A Y CYan, X., D. Broderick, et al. (2004) human RXRα LBD S L E A Y C K HYan, X., D. Broderick, et al. (2004) human RXRα LBD L E A Y C K H KYan, X., D. Broderick, et al. (2004) human RXRα LBD P G R F A K L LYan, X., D. Broderick, et al. (2004) human RXRα LBD F A K L L L R LYan, X., D. Broderick, et al. (2004) human RXRα LBD A K L L L R L PYan, X., D. Broderick, et al. (2004) human RXRα LBD L L R L P A L RYan, X., D. Broderick, et al. (2004) human RXRα LBD L P A L R S I GYan, X., D. Broderick, et al. (2004) human RXRα LBD A L R S I G L KYan, X., D. Broderick, et al. (2004) human RXRα LBD R S I G L K C LYan, X., D. Broderick, et al. (2004) human RXRα LBD S I G L K C L EYan, X., D. Broderick, et al. (2004) human RXRα LBD G L K C L E H LYan, X., D. Broderick, et al. (2004) human RXRα LBD K C L E H L F FYan, X., D. Broderick, et al. (2004) human RXRα LBD H L F F F K L IYan, X., D. Broderick, et al. (2004) human RXRα LBD P I D T F L M EYan, X., D. Broderick, et al. (2004) human RXRα LBD T F L M E M L EAnand, G. S., D. Law, et al. (2003) PKA - R1α A I S A E V Y TAnand, G. S., D. Law, et al. (2003) PKA - R1α D A A S Y V R KAnand, G. S., D. Law, et al. (2003) PKA - R1α K D Y K T M A AAnand, G. S., D. Law, et al. (2003) PKA - R1α T M A A L A K AAnand, G. S., D. Law, et al. (2003) PKA - R1α E K N V L F S HAnand, G. S., D. Law, et al. (2003) PKA - R1α K N V L F S H LAnand, G. S., D. Law, et al. (2003) PKA - R1α F S H L D D N EAnand, G. S., D. Law, et al. (2003) PKA - R1α L D D N E R S DAnand, G. S., D. Law, et al. (2003) PKA - R1α D D N E R S D IAnand, G. S., D. Law, et al. (2003) PKA - R1α R S D I F D A MAnand, G. S., D. Law, et al. (2003) PKA - R1α D E G D N F Y VAnand, G. S., D. Law, et al. (2003) PKA - R1α S F G E L A L IAnand, G. S., D. Law, et al. (2003) PKA - R1α E K A L I Y G TAnand, G. S., D. Law, et al. (2003) PKA - R1α N V K L W G I DAnand, G. S., D. Law, et al. (2003) PKA - R1α D S Y R R I L MAnand, G. S., D. Law, et al. (2003) PKA - R1α M G S T L R K RAnand, G. S., D. Law, et al. (2003) PKA - R1α G S T L R K R KAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit S V K E F L A KAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit A K E D L L K KAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit N T A Q D D Q FAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit T A Q L D Q F D
64
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Anand, G. S., D. Law, et al. (2003) PKA - C-Subunit L D Q F F R I KAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit G T G S G G R VAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit H K E S V N H YAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit K Q K V K K L KAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit Q K V V Q L K QAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit V K L K K I E HAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit T L N E P R I LAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit A V N F M F L VAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit A G G E R F S HAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit F S H L S R I GAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit I G R F Y E P HAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit H A R F Y A A QAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit L D L I L R D LAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit K P E N D L I DAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit N L L I S Q Q GAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit E I I L W K G YAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit A V D W F A L GAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit F P S H D S S DAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit H F S S L L K DAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit F S S D T K D LAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit G V D L I K R FAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit T T D W F A I YAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit D T S N D D D YAnand, G. S., D. Law, et al. (2003) PKA - C-Subunit T S N F D D Y EChik, J. K. and D. C. Schriemer (2003) PKA - C-Subunit T T A L V C D NChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin G S G L V K A GChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin R A V F P S I VChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin Q G V M V G M GChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin V M V G M G Q KChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin R G I L T L K YChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin G I L T L K Y PChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin W D D M E K I WChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin F Y N E L R V AChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin Y N E L R V A PChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin H P T L L T E AChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin K M T Q I M F EChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin T Q I M F E T FChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin Q I M F E T F NChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin V L S L Y A S GChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin G I V L D S G DChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin I Y E G Y A K PChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin I M R L D L A GChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin T D Y L M K I LChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin R S Y S F V T TChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin S Y S F V T T AChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin T T A E R E I VChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin A E R E I V R DChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin K L C Y V A L DChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin I G N E R F R CChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin Q P S F I G M EChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin N S I M K C D I
65
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Chik, J. K. and D. C. Schriemer (2003) rabbit muscle actin I M K C D I D IChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin Y A N N V M S GChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin K E I T A L A PChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin P S T M K I K IChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin Y S V W I G G SChik, J. K. and D. C. Schriemer (2003) rabbit muscle actin F Q Q M W I T KCravello, L., D. Lascoux, et al. (2003) PBP-2X* S P E F G T G TCravello, L., D. Lascoux, et al. (2003) PBP-2X* F G T D L A K ECravello, L., D. Lascoux, et al. (2003) PBP-2X* G T D L A K E ACravello, L., D. Lascoux, et al. (2003) PBP-2X* K E A K K V H QCravello, L., D. Lascoux, et al. (2003) PBP-2X* T V P A K R G TCravello, L., D. Lascoux, et al. (2003) PBP-2X* I A E D A T S YCravello, L., D. Lascoux, et al. (2003) PBP-2X* Y N V Y A V I DCravello, L., D. Lascoux, et al. (2003) PBP-2X* N V Y A V I D ECravello, L., D. Lascoux, et al. (2003) PBP-2X* V I D E N Y K SCravello, L., D. Lascoux, et al. (2003) PBP-2X* I D E N Y K S ACravello, L., D. Lascoux, et al. (2003) PBP-2X* E N Y K S A T GCravello, L., D. Lascoux, et al. (2003) PBP-2X* G K I L Y V E KCravello, L., D. Lascoux, et al. (2003) PBP-2X* K I L Y V E K TCravello, L., D. Lascoux, et al. (2003) PBP-2X* K T Q F N K V ACravello, L., D. Lascoux, et al. (2003) PBP-2X* K V A E V F H KCravello, L., D. Lascoux, et al. (2003) PBP-2X* V A E V F H K YCravello, L., D. Lascoux, et al. (2003) PBP-2X* Y L D M E E S YCravello, L., D. Lascoux, et al. (2003) PBP-2X* M E E S Y V R ECravello, L., D. Lascoux, et al. (2003) PBP-2X* E E S Y V R E QCravello, L., D. Lascoux, et al. (2003) PBP-2X* Q P N L K Q V SCravello, L., D. Lascoux, et al. (2003) PBP-2X* A K G N G I T YCravello, L., D. Lascoux, et al. (2003) PBP-2X* G N G I T Y A NCravello, L., D. Lascoux, et al. (2003) PBP-2X* Y A N M M S I KCravello, L., D. Lascoux, et al. (2003) PBP-2X* K K E L E A A ECravello, L., D. Lascoux, et al. (2003) PBP-2X* K E L E A A E VCravello, L., D. Lascoux, et al. (2003) PBP-2X* E L E A A E V KCravello, L., D. Lascoux, et al. (2003) PBP-2X* L E A A E V K GCravello, L., D. Lascoux, et al. (2003) PBP-2X* E A A E V K G ICravello, L., D. Lascoux, et al. (2003) PBP-2X* K G I D F T T SCravello, L., D. Lascoux, et al. (2003) PBP-2X* G I D F T T S PCravello, L., D. Lascoux, et al. (2003) PBP-2X* S Y P N G Q F ACravello, L., D. Lascoux, et al. (2003) PBP-2X* Y P N G Q F A SCravello, L., D. Lascoux, et al. (2003) PBP-2X* P N G Q F A S SCravello, L., D. Lascoux, et al. (2003) PBP-2X* A S S F I G L ACravello, L., D. Lascoux, et al. (2003) PBP-2X* S F I G L A Q LCravello, L., D. Lascoux, et al. (2003) PBP-2X* I G L Q A L H ECravello, L., D. Lascoux, et al. (2003) PBP-2X* L A Q L H E N ECravello, L., D. Lascoux, et al. (2003) PBP-2X* Q L H E N E D GCravello, L., D. Lascoux, et al. (2003) PBP-2X* K S L L G T S GCravello, L., D. Lascoux, et al. (2003) PBP-2X* M E S S L N L ICravello, L., D. Lascoux, et al. (2003) PBP-2X* N S I L A G T DCravello, L., D. Lascoux, et al. (2003) PBP-2X* L A G T D G I ICravello, L., D. Lascoux, et al. (2003) PBP-2X* G I I T Y E K DCravello, L., D. Lascoux, et al. (2003) PBP-2X* P G T E Q V S QCravello, L., D. Lascoux, et al. (2003) PBP-2X* G T E Q V S Q R
66
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Cravello, L., D. Lascoux, et al. (2003) PBP-2X* M D G K D V Y TCravello, L., D. Lascoux, et al. (2003) PBP-2X* D G K D V Y T TCravello, L., D. Lascoux, et al. (2003) PBP-2X* K D V Y T T I SCravello, L., D. Lascoux, et al. (2003) PBP-2X* L Q S E M E T QCravello, L., D. Lascoux, et al. (2003) PBP-2X* Q S E M E T Q MCravello, L., D. Lascoux, et al. (2003) PBP-2X* Q M D A F Q F KCravello, L., D. Lascoux, et al. (2003) PBP-2X* F Q F K V K G KCravello, L., D. Lascoux, et al. (2003) PBP-2X* T A T L V S A KCravello, L., D. Lascoux, et al. (2003) PBP-2X* S A K T G E I LCravello, L., D. Lascoux, et al. (2003) PBP-2X* G E I L A T T QCravello, L., D. Lascoux, et al. (2003) PBP-2X* L A T T Q R P TCravello, L., D. Lascoux, et al. (2003) PBP-2X* T Q R P T F D ACravello, L., D. Lascoux, et al. (2003) PBP-2X* R P T F D A D TCravello, L., D. Lascoux, et al. (2003) PBP-2X* T K E G I T E DCravello, L., D. Lascoux, et al. (2003) PBP-2X* I T E D F V W RCravello, L., D. Lascoux, et al. (2003) PBP-2X* T E D F V W R DCravello, L., D. Lascoux, et al. (2003) PBP-2X* R D I L Y Q S NCravello, L., D. Lascoux, et al. (2003) PBP-2X* G S T M K V M MCravello, L., D. Lascoux, et al. (2003) PBP-2X* M K V M M L A ACravello, L., D. Lascoux, et al. (2003) PBP-2X* K V M M L A A ACravello, L., D. Lascoux, et al. (2003) PBP-2X* V M M L A A A ICravello, L., D. Lascoux, et al. (2003) PBP-2X* I D N N T F P GCravello, L., D. Lascoux, et al. (2003) PBP-2X* G E V F N S S ECravello, L., D. Lascoux, et al. (2003) PBP-2X* N S S E L K I ACravello, L., D. Lascoux, et al. (2003) PBP-2X* I A D A T I R DCravello, L., D. Lascoux, et al. (2003) PBP-2X* T I R D W D V NCravello, L., D. Lascoux, et al. (2003) PBP-2X* A T I R D W D VCravello, L., D. Lascoux, et al. (2003) PBP-2X* I R D W D V N ECravello, L., D. Lascoux, et al. (2003) PBP-2X* E G L T G G R MCravello, L., D. Lascoux, et al. (2003) PBP-2X* G G R M M T F SCravello, L., D. Lascoux, et al. (2003) PBP-2X* G R M M T F S QCravello, L., D. Lascoux, et al. (2003) PBP-2X* M M T F S Q G FCravello, L., D. Lascoux, et al. (2003) PBP-2X* G M T L L E Q KCravello, L., D. Lascoux, et al. (2003) PBP-2X* D A T W L D Y LCravello, L., D. Lascoux, et al. (2003) PBP-2X* A T W L D Y L NCravello, L., D. Lascoux, et al. (2003) PBP-2X* F G V P T R F GCravello, L., D. Lascoux, et al. (2003) PBP-2X* P T R F G L T DCravello, L., D. Lascoux, et al. (2003) PBP-2X* L T D E Y A G QCravello, L., D. Lascoux, et al. (2003) PBP-2X* A D N I V N I ACravello, L., D. Lascoux, et al. (2003) PBP-2X* V N I A Q S S FCravello, L., D. Lascoux, et al. (2003) PBP-2X* A F T A I A N DCravello, L., D. Lascoux, et al. (2003) PBP-2X* A N D G V M L ECravello, L., D. Lascoux, et al. (2003) PBP-2X* N D G V M L E PCravello, L., D. Lascoux, et al. (2003) PBP-2X* F I S A I Y D PCravello, L., D. Lascoux, et al. (2003) PBP-2X* I Y D P N D Q TCravello, L., D. Lascoux, et al. (2003) PBP-2X* D Q T A R K S QCravello, L., D. Lascoux, et al. (2003) PBP-2X* Q T A R K S Q KCravello, L., D. Lascoux, et al. (2003) PBP-2X* R K S Q K E I VCravello, L., D. Lascoux, et al. (2003) PBP-2X* K S Q K E I V GCravello, L., D. Lascoux, et al. (2003) PBP-2X* I V G N P V S KCravello, L., D. Lascoux, et al. (2003) PBP-2X* N P V S K D A A
67
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Cravello, L., D. Lascoux, et al. (2003) PBP-2X* A A S L T R T NCravello, L., D. Lascoux, et al. (2003) PBP-2X* S L T R T N M VCravello, L., D. Lascoux, et al. (2003) PBP-2X* T N M V L V G TCravello, L., D. Lascoux, et al. (2003) PBP-2X* N M V L V G T DCravello, L., D. Lascoux, et al. (2003) PBP-2X* D P V Y G T M YCravello, L., D. Lascoux, et al. (2003) PBP-2X* Y G T M Y N H SCravello, L., D. Lascoux, et al. (2003) PBP-2X* G T M Y N H S TCravello, L., D. Lascoux, et al. (2003) PBP-2X* P G Q N V A L KCravello, L., D. Lascoux, et al. (2003) PBP-2X* G T A Q I A D ECravello, L., D. Lascoux, et al. (2003) PBP-2X* Q I A D E K N GCravello, L., D. Lascoux, et al. (2003) PBP-2X* G G Y L V G L TCravello, L., D. Lascoux, et al. (2003) PBP-2X* V G L T D Y I FCravello, L., D. Lascoux, et al. (2003) PBP-2X* D Y I F S A V SCravello, L., D. Lascoux, et al. (2003) PBP-2X* Y I F S A V S MCravello, L., D. Lascoux, et al. (2003) PBP-2X* I F S A V S M SCravello, L., D. Lascoux, et al. (2003) PBP-2X* A V S M S P A ECravello, L., D. Lascoux, et al. (2003) PBP-2X* N P D F I L Y VCravello, L., D. Lascoux, et al. (2003) PBP-2X* D F I L Y V T VCravello, L., D. Lascoux, et al. (2003) PBP-2X* I L Y V T V Q QCravello, L., D. Lascoux, et al. (2003) PBP-2X* V T V Q Q P E HCravello, L., D. Lascoux, et al. (2003) PBP-2X* Y S G I Q L G ECravello, L., D. Lascoux, et al. (2003) PBP-2X* I Q L G E F A NCravello, L., D. Lascoux, et al. (2003) PBP-2X* L G E F A N P ICravello, L., D. Lascoux, et al. (2003) PBP-2X* A N P I L E R ACravello, L., D. Lascoux, et al. (2003) PBP-2X* N P I L E R A SCravello, L., D. Lascoux, et al. (2003) PBP-2X* A S A M K D S LCravello, L., D. Lascoux, et al. (2003) PBP-2X* S L N L Q T T ACravello, L., D. Lascoux, et al. (2003) PBP-2X* L N L Q T T A KCravello, L., D. Lascoux, et al. (2003) PBP-2X* A K A L E Q V SCravello, L., D. Lascoux, et al. (2003) PBP-2X* P G D L A E E LCravello, L., D. Lascoux, et al. (2003) PBP-2X* G D L A E E L RCravello, L., D. Lascoux, et al. (2003) PBP-2X* L V Q P I V V GCravello, L., D. Lascoux, et al. (2003) PBP-2X* V G T G T K I KCravello, L., D. Lascoux, et al. (2003) PBP-2X* T G T K I K N SCravello, L., D. Lascoux, et al. (2003) PBP-2X* T K I K N S S ACravello, L., D. Lascoux, et al. (2003) PBP-2X* A E E G K N L ACravello, L., D. Lascoux, et al. (2003) PBP-2X* V L I L S D K ACravello, L., D. Lascoux, et al. (2003) PBP-2X* E E V P D M Y GCravello, L., D. Lascoux, et al. (2003) PBP-2X* P D M Y G W T KCravello, L., D. Lascoux, et al. (2003) PBP-2X* G W T K E T A ECravello, L., D. Lascoux, et al. (2003) PBP-2X* T A E T L A K WCravello, L., D. Lascoux, et al. (2003) PBP-2X* A E T L A K W LCravello, L., D. Lascoux, et al. (2003) PBP-2X* K W L N I E L ECravello, L., D. Lascoux, et al. (2003) PBP-2X* N I E L E F Q GCravello, L., D. Lascoux, et al. (2003) PBP-2X* D V R A N T A ICravello, L., D. Lascoux, et al. (2003) PBP-2X* A I K D I K K ICravello, L., D. Lascoux, et al. (2003) PBP-2X* T L T L G DRist, W., T. J. Jorgensen, et al. (2003) σ 32 M Q S L A L A PRist, W., T. J. Jorgensen, et al. (2003) σ 32 V G N L D S Y IRist, W., T. J. Jorgensen, et al. (2003) σ 32 N L D S Y I R ARist, W., T. J. Jorgensen, et al. (2003) σ 32 W P M L S A D E
68
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Rist, W., T. J. Jorgensen, et al. (2003) σ 32 D L E A A K T LRist, W., T. J. Jorgensen, et al. (2003) σ 32 A A K T L I L SRist, W., T. J. Jorgensen, et al. (2003) σ 32 H L R F V V H IRist, W., T. J. Jorgensen, et al. (2003) σ 32 Q A D L I Q E GRist, W., T. J. Jorgensen, et al. (2003) σ 32 N I G L M K A VRist, W., T. J. Jorgensen, et al. (2003) σ 32 L M K A V R R FRist, W., T. J. Jorgensen, et al. (2003) σ 32 P E V G V R L VRist, W., T. J. Jorgensen, et al. (2003) σ 32 G V R L V S F ARist, W., T. J. Jorgensen, et al. (2003) σ 32 R L V S F A V HRist, W., T. J. Jorgensen, et al. (2003) σ 32 L V S F A V H WRist, W., T. J. Jorgensen, et al. (2003) σ 32 E I H E Y V L RRist, W., T. J. Jorgensen, et al. (2003) σ 32 V L R N W R I VRist, W., T. J. Jorgensen, et al. (2003) σ 32 K L F F N L R KRist, W., T. J. Jorgensen, et al. (2003) σ 32 W F N Q D E V ERist, W., T. J. Jorgensen, et al. (2003) σ 32 N Q D E V E M VRist, W., T. J. Jorgensen, et al. (2003) σ 32 D E V E M V A RRist, W., T. J. Jorgensen, et al. (2003) σ 32 E V E M V A R ERist, W., T. J. Jorgensen, et al. (2003) σ 32 E S R M A A Q DRist, W., T. J. Jorgensen, et al. (2003) σ 32 Q D M T F D L SRist, W., T. J. Jorgensen, et al. (2003) σ 32 D M T F D L S SRist, W., T. J. Jorgensen, et al. (2003) σ 32 A P V L Y L Q DRist, W., T. J. Jorgensen, et al. (2003) σ 32 K S S N F A D GRist, W., T. J. Jorgensen, et al. (2003) σ 32 S S N F A D G IRist, W., T. J. Jorgensen, et al. (2003) σ 32 I E D D N W E ERist, W., T. J. Jorgensen, et al. (2003) σ 32 R S Q D I I R ARist, W., T. J. Jorgensen, et al. (2003) σ 32 R A R W L D E DRist, W., T. J. Jorgensen, et al. (2003) σ 32 K S T L Q E L ARist, W., T. J. Jorgensen, et al. (2003) σ 32 L Q E L A D R YRist, W., T. J. Jorgensen, et al. (2003) σ 32 Q E L A D R Y GRist, W., T. J. Jorgensen, et al. (2003) σ 32 Y G V S A E R VRist, W., T. J. Jorgensen, et al. (2003) σ 32 Q L E K N A M KWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 M S I V RWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 F A D L W A D PWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 P F D T F R S IWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 F D T F R S I VWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 E T A A F A N AWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 N A R M D W K EWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 A R M D W K E TWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 A H V F K A D LWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 G N V L V V S GWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 N D K W H R V EWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 S G K F V R R FWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 L L E D A K V EWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 K V E E V K A GWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 N G V L T V T VWintrode, P. L., K. L. Friedrich, et al. (2003) HSP16.9 E V K A I Q I SHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA M D V T I QHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA P S R L F D Q FHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA D Q F F G E G LHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA E G L F E Y D LHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA Y D L L P F L S
69
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Hasan, A., D. L. Smith, et al. (2002) α-Crystallin αA R Q S L P R T VHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA R T V L D S G IHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA S G I S G V R SHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA R D K F V I F LHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA F V I F L D V KHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA I F L D V K H FHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA P E D L T V K VHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA Q D D F V E I HHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA E R Q D D H G YHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA D D H G Y I S RHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA V D Q S A L S CHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA Q S A L S C S LHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA S C S L S A D GHasan, A., D. L. Smith, et al. (2002) α-Crystallin αA D G M L T F C GHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ M D I A I HHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ P S R L F D Q FHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ R L F D Q F F GHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ F D Q F F G E HHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ E S D L F P T SHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ P P S F L R A PHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ S W F D T G L SHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ G L S E M R L EHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ K D R F S V N LHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ S V N L D V K HHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ P E E L K V K VHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ D V I E V H G KHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ I T S S L S S DHasan, A., D. L. Smith, et al. (2002) α-Crystallin αΒ D G V L T V N GYan, X., H. Zhang, et al. (2002) rhM-CSFß S G H L Q S L QYan, X., H. Zhang, et al. (2002) rhM-CSFß L Q S L Q R L IYan, X., H. Zhang, et al. (2002) rhM-CSFß D S Q M E T S CYan, X., H. Zhang, et al. (2002) rhM-CSFß I T F E F V D QYan, X., H. Zhang, et al. (2002) rhM-CSFß K A F L L V Q DYan, X., H. Zhang, et al. (2002) rhM-CSFß D I M E D T M RYan, X., H. Zhang, et al. (2002) rhM-CSFß T M R F R D N TYan, X., H. Zhang, et al. (2002) rhM-CSFß T P N A I A I VYan, X., H. Zhang, et al. (2002) rhM-CSFß N A I A I V Q KYan, X., H. Zhang, et al. (2002) rhM-CSFß I V Q L Q E L SYan, X., H. Zhang, et al. (2002) rhM-CSFß Q L Q E L S L RYan, X., H. Zhang, et al. (2002) rhM-CSFß E L S L R L K SYan, X., H. Zhang, et al. (2002) rhM-CSFß C V R T F Y E TYan, X., H. Zhang, et al. (2002) rhM-CSFß V R T F Y E T PYan, X., H. Zhang, et al. (2002) rhM-CSFß P L Q L L E K VYan, X., H. Zhang, et al. (2002) rhM-CSFß K N L L D K D WYan, X., H. Zhang, et al. (2002) rhM-CSFß W N I F S K N CYan, X., H. Zhang, et al. (2002) rhM-CSFß C S S Q D V V TYan, X., H. Zhang, et al. (2002) rhM-CSFß C N C L Y P K AYan, X., H. Zhang, et al. (2002) rhM-CSFß V A Q L T W E DYan, X., H. Zhang, et al. (2002) rhM-CSFß L T W E D S E GYan, X., H. Zhang, et al. (2002) rhM-CSFß S S L L P G E QYan, X., H. Zhang, et al. (2002) rhM-CSFß H T V D P G S A
70
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Hughes, C. A., J. G. Mandell, et al. (2001) CheB M S K I RHughes, C. A., J. G. Mandell, et al. (2001) CheB D S A L M R Q IHughes, C. A., J. G. Mandell, et al. (2001) CheB I M T E I I N SHughes, C. A., J. G. Mandell, et al. (2001) CheB D M E M V A T AHughes, C. A., J. G. Mandell, et al. (2001) CheB P L V A R D L IHughes, C. A., J. G. Mandell, et al. (2001) CheB V A R D L I K KHughes, C. A., J. G. Mandell, et al. (2001) CheB V L T L D V E MHughes, C. A., J. G. Mandell, et al. (2001) CheB L T L D V E M PHughes, C. A., J. G. Mandell, et al. (2001) CheB D G L D F L E KHughes, C. A., J. G. Mandell, et al. (2001) CheB G L D F L E K LHughes, C. A., J. G. Mandell, et al. (2001) CheB L D F L E K L MHughes, C. A., J. G. Mandell, et al. (2001) CheB L E K L M R L RHughes, C. A., J. G. Mandell, et al. (2001) CheB P V V M V S S LHughes, C. A., J. G. Mandell, et al. (2001) CheB A L E L G A I DHughes, C. A., J. G. Mandell, et al. (2001) CheB A I D F V T K PHughes, C. A., J. G. Mandell, et al. (2001) CheB E G M L A Y S EHughes, C. A., J. G. Mandell, et al. (2001) CheB G M L A Y S E MHughes, C. A., J. G. Mandell, et al. (2001) CheB Y S E M I A E KHughes, C. A., J. G. Mandell, et al. (2001) CheB P T T L K A G PHughes, C. A., J. G. Mandell, et al. (2001) CheB S E K L I A I GHughes, C. A., J. G. Mandell, et al. (2001) CheB G G T E A I R HHughes, C. A., J. G. Mandell, et al. (2001) CheB G T E A I R H VHughes, C. A., J. G. Mandell, et al. (2001) CheB P L S S P A V IHughes, C. A., J. G. Mandell, et al. (2001) CheB P A V I I T Q HHughes, C. A., J. G. Mandell, et al. (2001) CheB P P G F T R S FHughes, C. A., J. G. Mandell, et al. (2001) CheB A E R L N K L CHughes, C. A., J. G. Mandell, et al. (2001) CheB V L P G H A Y IHughes, C. A., J. G. Mandell, et al. (2001) CheB P G H A Y I A PHughes, C. A., J. G. Mandell, et al. (2001) CheB G H A Y I A P GHughes, C. A., J. G. Mandell, et al. (2001) CheB H M E L A R S GHughes, C. A., J. G. Mandell, et al. (2001) CheB S G A N Y Q I KHughes, C. A., J. G. Mandell, et al. (2001) CheB P S V D V L F HHughes, C. A., J. G. Mandell, et al. (2001) CheB V D V L F H S VHughes, C. A., J. G. Mandell, et al. (2001) CheB D V L F H S V AHughes, C. A., J. G. Mandell, et al. (2001) CheB G V I L T G M GHughes, C. A., J. G. Mandell, et al. (2001) CheB E A S C V V F GHughes, C. A., J. G. Mandell, et al. (2001) CheB M P R E A I N MHughes, C. A., J. G. Mandell, et al. (2001) CheB G V S E V V D LHughes, C. A., J. G. Mandell, et al. (2001) CheB V S E V V D L SHughes, C. A., J. G. Mandell, et al. (2001) CheB Q M L A K I S AWang, L., L. C. Lane, et al. (2001) BMV A I A G Y S I SWang, L., L. C. Lane, et al. (2001) BMV A S S D A I T AWang, L., L. C. Lane, et al. (2001) BMV S D A I T A K AWang, L., L. C. Lane, et al. (2001) BMV A T N A M S I TWang, L., L. C. Lane, et al. (2001) BMV N A M S I T L PWang, L., L. C. Lane, et al. (2001) BMV G R V L L W L GWang, L., L. C. Lane, et al. (2001) BMV R V L L W L G LWang, L., L. C. Lane, et al. (2001) BMV L L W L G L L PWang, L., L. C. Lane, et al. (2001) BMV L G L L P S V AWang, L., L. C. Lane, et al. (2001) BMV R I K A C V A EWang, L., L. C. Lane, et al. (2001) BMV K A C V A E K Q
71
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Wang, L., L. C. Lane, et al. (2001) BMV A C V A E K Q AWang, L., L. C. Lane, et al. (2001) BMV A E A A F Q V AWang, L., L. C. Lane, et al. (2001) BMV E A A F Q V A LWang, L., L. C. Lane, et al. (2001) BMV A F Q V A L A VWang, L., L. C. Lane, et al. (2001) BMV F Q V A L A V AWang, L., L. C. Lane, et al. (2001) BMV Q V A L A V A DWang, L., L. C. Lane, et al. (2001) BMV S S K E V V A AWang, L., L. C. Lane, et al. (2001) BMV V A A M Y T D AWang, L., L. C. Lane, et al. (2001) BMV Y T D A F R G AWang, L., L. C. Lane, et al. (2001) BMV G D L L N L Q IWang, L., L. C. Lane, et al. (2001) BMV L L N L Q I Y LWang, L., L. C. Lane, et al. (2001) BMV Q I Y L Y A S EWang, L., L. C. Lane, et al. (2001) BMV P A K A V V H LWang, L., L. C. Lane, et al. (2001) BMV V V H L E V E HWang, L., L. C. Lane, et al. (2001) BMV F D D F F T P VChen, J. and D. L. Smith (2000) GroEL R G V N V L A DChen, J. and D. L. Smith (2000) GroEL K G R N V V L DChen, J. and D. L. Smith (2000) GroEL D G V S V A R EChen, J. and D. L. Smith (2000) GroEL E I E L E D K FChen, J. and D. L. Smith (2000) GroEL G A Q M V K E VChen, J. and D. L. Smith (2000) GroEL A T V L A Q A IChen, J. and D. L. Smith (2000) GroEL V T A A V E E LChen, J. and D. L. Smith (2000) GroEL E L K A L S V PChen, J. and D. L. Smith (2000) GroEL D S K A I A Q VChen, J. and D. L. Smith (2000) GroEL V G K L I A E AChen, J. and D. L. Smith (2000) GroEL A E A M D K V GChen, J. and D. L. Smith (2000) GroEL Q D E L D V V EChen, J. and D. L. Smith (2000) GroEL D E L D V V E GChen, J. and D. L. Smith (2000) GroEL E G M Q F D R GChen, J. and D. L. Smith (2000) GroEL S P Y F I N K PChen, J. and D. L. Smith (2000) GroEL N K P E T G A VChen, J. and D. L. Smith (2000) GroEL G A V E L E S PChen, J. and D. L. Smith (2000) GroEL P F I L L A D KChen, J. and D. L. Smith (2000) GroEL F I L L A D K KChen, J. and D. L. Smith (2000) GroEL R E M L P V L EChen, J. and D. L. Smith (2000) GroEL L P V L E A V AChen, J. and D. L. Smith (2000) GroEL K P L L I I A EChen, J. and D. L. Smith (2000) GroEL I I A E D V E GChen, J. and D. L. Smith (2000) GroEL G E A L A T L VChen, J. and D. L. Smith (2000) GroEL L A T L V V N TChen, J. and D. L. Smith (2000) GroEL T M R G I V K VChen, J. and D. L. Smith (2000) GroEL M L Q D I A T LChen, J. and D. L. Smith (2000) GroEL I S E E I G M EChen, J. and D. L. Smith (2000) GroEL E E I G M E L EChen, J. and D. L. Smith (2000) GroEL K A T L E D L GChen, J. and D. L. Smith (2000) GroEL D T T T I I D GChen, J. and D. L. Smith (2000) GroEL Q I E E A T S DChen, J. and D. L. Smith (2000) GroEL K L Q E R V A KChen, J. and D. L. Smith (2000) GroEL G G V A V I K VChen, J. and D. L. Smith (2000) GroEL A A T E V E M KChen, J. and D. L. Smith (2000) GroEL E D A L H A T R
72
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Chen, J. and D. L. Smith (2000) GroEL A V E E G V V AChen, J. and D. L. Smith (2000) GroEL G V A L I R V AChen, J. and D. L. Smith (2000) GroEL A S K L A D L RChen, J. and D. L. Smith (2000) GroEL L A D L R G Q NChen, J. and D. L. Smith (2000) GroEL K V A L R A M EChen, J. and D. L. Smith (2000) GroEL P L R Q I V L NChen, J. and D. L. Smith (2000) GroEL A T E E Y G N MChen, J. and D. L. Smith (2000) GroEL Y G N M I D M GChen, J. and D. L. Smith (2000) GroEL M I D M G I L DChen, J. and D. L. Smith (2000) GroEL S A L Q Y A A SChen, J. and D. L. Smith (2000) GroEL V A G L M I T TChen, J. and D. L. Smith (2000) GroEL T E C M V T D LEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) G S E D I I V VEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) I V V A L Y D YEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) V V A L Y D Y EEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) H H E D L S F QEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) H E D L S F Q KEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) D L S F Q K G DEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) G D Q M V V L EEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) S G E W W K A REngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) I P S I Y V A REngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) P S I Y V A R VEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) S I Y V A R V DEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) Y V A R V D S LEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) V D S L E T E EEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) S L E T E E W FEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) T E E W F F K GEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) F F K G I S R KEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) A R G N M L G SEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) N M L G S F M IEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) L G S F M I R DEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) G S F M I R D SEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) S Y S L S V R DEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) D N G G F Y I SEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) N G G F Y I S PEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) P R S T F S T LEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) F S T L Q E L VEngen, J. R., T. E. Smithgall, et al. (1999) Hck SH(3 + 2) N D G L C Q K LWang, F., W. Li, et al. (1999) cNTnC I Y K A A V E QWang, F., W. Li, et al. (1999) cNTnC A V E Q L T E EWang, F., W. Li, et al. (1999) cNTnC K A A F D I F VWang, F., W. Li, et al. (1999) cNTnC F D I F V L G AWang, F., W. Li, et al. (1999) cNTnC I F V L G A E DWang, F., W. Li, et al. (1999) cNTnC E D G C I S T KWang, F., W. Li, et al. (1999) cNTnC V M R M L G Q NWang, F., W. Li, et al. (1999) cNTnC T P E E L Q E MWang, F., W. Li, et al. (1999) cNTnC P E E L Q E M IWang, F., W. Li, et al. (1999) cNTnC I D E V D E D GResing, K. A. and N. G. Ahn (1998) human MKK1 K K K P T P I QResing, K. A. and N. G. Ahn (1998) human MKK1 E T N L E A L QResing, K. A. and N. G. Ahn (1998) human MKK1 K L E E L E L D
73
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Resing, K. A. and N. G. Ahn (1998) human MKK1 R L E A F L T QResing, K. A. and N. G. Ahn (1998) human MKK1 L E A F L T Q KResing, K. A. and N. G. Ahn (1998) human MKK1 K D D D F E K IResing, K. A. and N. G. Ahn (1998) human MKK1 D D D F E K I SResing, K. A. and N. G. Ahn (1998) human MKK1 K I S E L G A GResing, K. A. and N. G. Ahn (1998) human MKK1 G V V F K V S HResing, K. A. and N. G. Ahn (1998) human MKK1 G L V M A R K LResing, K. A. and N. G. Ahn (1998) human MKK1 L I H L E I K PResing, K. A. and N. G. Ahn (1998) human MKK1 I I R E L Q V LResing, K. A. and N. G. Ahn (1998) human MKK1 I V G F Y G A FResing, K. A. and N. G. Ahn (1998) human MKK1 Y G A F Y S D GResing, K. A. and N. G. Ahn (1998) human MKK1 G E I S I C M EResing, K. A. and N. G. Ahn (1998) human MKK1 I S I C M E H MResing, K. A. and N. G. Ahn (1998) human MKK1 G G S L D Q V LResing, K. A. and N. G. Ahn (1998) human MKK1 V S I A V I K GResing, K. A. and N. G. Ahn (1998) human MKK1 L T Y L R E K HResing, K. A. and N. G. Ahn (1998) human MKK1 K P S N I L V NResing, K. A. and N. G. Ahn (1998) human MKK1 E I K L C D V GResing, K. A. and N. G. Ahn (1998) human MKK1 S G Q L I D S MResing, K. A. and N. G. Ahn (1998) human MKK1 L I D S M A N SResing, K. A. and N. G. Ahn (1998) human MKK1 M A N S F V G TResing, K. A. and N. G. Ahn (1998) human MKK1 A N S F V G T RResing, K. A. and N. G. Ahn (1998) human MKK1 G T H Y S V Q SResing, K. A. and N. G. Ahn (1998) human MKK1 W S M G L S L VResing, K. A. and N. G. Ahn (1998) human MKK1 G L S L V E M AResing, K. A. and N. G. Ahn (1998) human MKK1 D A K E L E L MResing, K. A. and N. G. Ahn (1998) human MKK1 E L E L M F G CResing, K. A. and N. G. Ahn (1998) human MKK1 E L M F G C Q VResing, K. A. and N. G. Ahn (1998) human MKK1 F G C Q V E G DResing, K. A. and N. G. Ahn (1998) human MKK1 E G D A A E T PResing, K. A. and N. G. Ahn (1998) human MKK1 M A I F E L L DResing, K. A. and N. G. Ahn (1998) human MKK1 E L L D Y I V NResing, K. A. and N. G. Ahn (1998) human MKK1 S G V F S L E FResing, K. A. and N. G. Ahn (1998) human MKK1 F S L E F Q D FResing, K. A. and N. G. Ahn (1998) human MKK1 M V H A F I K RResing, K. A. and N. G. Ahn (1998) human MKK1 D A E E V D F AResing, K. A. and N. G. Ahn (1998) human MKK1 A G W L C S T IResing, K. A. and N. G. Ahn (1998) human MKK1 L C S T I G L NNeubert, T. A., K. A. Walsh, et al. (1997) recoverin L S K E I L E ENeubert, T. A., K. A. Walsh, et al. (1997) recoverin I L E E L Q L NNeubert, T. A., K. A. Walsh, et al. (1997) recoverin E L Q L N T K FNeubert, T. A., K. A. Walsh, et al. (1997) recoverin E E E L S S W YNeubert, T. A., K. A. Walsh, et al. (1997) recoverin L S S W Y Q S FNeubert, T. A., K. A. Walsh, et al. (1997) recoverin Y Q S F L K E CNeubert, T. A., K. A. Walsh, et al. (1997) recoverin Q S F L K E C PNeubert, T. A., K. A. Walsh, et al. (1997) recoverin Q T I Y S K F FNeubert, T. A., K. A. Walsh, et al. (1997) recoverin Y S K F F P E ANeubert, T. A., K. A. Walsh, et al. (1997) recoverin D P K A Y A Q HNeubert, T. A., K. A. Walsh, et al. (1997) recoverin Q H V F R S F DNeubert, T. A., K. A. Walsh, et al. (1997) recoverin D G T L D F K ENeubert, T. A., K. A. Walsh, et al. (1997) recoverin D F K E Y V I A
74
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Neubert, T. A., K. A. Walsh, et al. (1997) recoverin V I A L H M T SNeubert, T. A., K. A. Walsh, et al. (1997) recoverin L E W A F S L YNeubert, T. A., K. A. Walsh, et al. (1997) recoverin A F S L Y D V DNeubert, T. A., K. A. Walsh, et al. (1997) recoverin N E V L E I V TNeubert, T. A., K. A. Walsh, et al. (1997) recoverin E I V T A I F KNeubert, T. A., K. A. Walsh, et al. (1997) recoverin T A I F K M I SNeubert, T. A., K. A. Walsh, et al. (1997) recoverin I W G F F G K KNeubert, T. A., K. A. Walsh, et al. (1997) recoverin T E K E F I E GNeubert, T. A., K. A. Walsh, et al. (1997) recoverin A N K E I L R LNeubert, T. A., K. A. Walsh, et al. (1997) recoverin I L R L I Q F EWang, F., J. S. Blanchard, et al. (1997) DHPR H D A N I R V AWang, F., J. S. Blanchard, et al. (1997) DHPR A N I R V A I AWang, F., J. S. Blanchard, et al. (1997) DHPR I Q A A L A L EWang, F., J. S. Blanchard, et al. (1997) DHPR Q A A L A L E GWang, F., J. S. Blanchard, et al. (1997) DHPR G A A L E R E GWang, F., J. S. Blanchard, et al. (1997) DHPR Q S S L D A V KWang, F., J. S. Blanchard, et al. (1997) DHPR K D D F D V F IWang, F., J. S. Blanchard, et al. (1997) DHPR F D V F I D F TWang, F., J. S. Blanchard, et al. (1997) DHPR H L A F C R Q HWang, F., J. S. Blanchard, et al. (1997) DHPR T T G F D E A GWang, F., J. S. Blanchard, et al. (1997) DHPR A A A D I A I VWang, F., J. S. Blanchard, et al. (1997) DHPR A I V F A A N FWang, F., J. S. Blanchard, et al. (1997) DHPR A N F S V G V NWang, F., J. S. Blanchard, et al. (1997) DHPR M L K L L E K AWang, F., J. S. Blanchard, et al. (1997) DHPR D Y T D I E I IWang, F., J. S. Blanchard, et al. (1997) DHPR G T A L A M G EWang, F., J. S. Blanchard, et al. (1997) DHPR G E A I A H A LWang, F., J. S. Blanchard, et al. (1997) DHPR D C A V Y S R EWang, F., J. S. Blanchard, et al. (1997) DHPR T I G F A T V RWang, F., J. S. Blanchard, et al. (1997) DHPR H T A M F A D IWang, F., J. S. Blanchard, et al. (1997) DHPR G E R L E I T HWang, F., J. S. Blanchard, et al. (1997) DHPR F A N G A V R SWang, F., J. S. Blanchard, et al. (1997) DHPR R S A L W L S GWang, F., J. S. Blanchard, et al. (1997) DHPR E S G L F D M RWang, F., J. S. Blanchard, et al. (1997) DHPR D M R D V L D LWang, F., J. S. Blanchard, et al. (1997) DHPR V L D L N N LDharmasiri, K. and D. L. Smith (1996) horse heart cyt c K K I F V Q K CDharmasiri, K. and D. L. Smith (1996) horse heart cyt c H T V E K G G KDharmasiri, K. and D. L. Smith (1996) horse heart cyt c G P N L H G L FDharmasiri, K. and D. L. Smith (1996) horse heart cyt c H G L F G R K TDharmasiri, K. and D. L. Smith (1996) horse heart cyt c A P G F T Y T DDharmasiri, K. and D. L. Smith (1996) horse heart cyt c P G F T Y T D ADharmasiri, K. and D. L. Smith (1996) horse heart cyt c E E T L M E Y LDharmasiri, K. and D. L. Smith (1996) horse heart cyt c E T L M E Y L EDharmasiri, K. and D. L. Smith (1996) horse heart cyt c T L M E Y L E NDharmasiri, K. and D. L. Smith (1996) horse heart cyt c L M E Y L E N PDharmasiri, K. and D. L. Smith (1996) horse heart cyt c G T K M I F A GDharmasiri, K. and D. L. Smith (1996) horse heart cyt c T K M I F A G IDharmasiri, K. and D. L. Smith (1996) horse heart cyt c K M I F A G I KDharmasiri, K. and D. L. Smith (1996) horse heart cyt c F A G I K K T EDharmasiri, K. and D. L. Smith (1996) horse heart cyt c R E D L I A Y L
75
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Dharmasiri, K. and D. L. Smith (1996) horse heart cyt c E D L I A Y L KDharmasiri, K. and D. L. Smith (1996) horse heart cyt c D L I A Y L K KDharmasiri, K. and D. L. Smith (1996) horse heart cyt c L I A Y L K K AZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase H P A L T P E QZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase E L S D I A H RZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase K G I L A A D EZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase G S I A K R L QZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase I G T E N T E EZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase N T E E N R R FZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase N R R F Y R Q LZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase R G L L L T A DZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase Q L L L T A D DZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase L L L T A D D RZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase L T A D D R V NZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase T A D D R V N PZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase P C I G G V I LZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase G V I L F H E TZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase H E T L Y Q K AZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase G V V G I K V DZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase L D G L S E R CZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase D G L S E R C AZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase D G A D F A K WZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase R C V L K I G EZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase T P S A L A I MZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase P S A L A I M EZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase S A L A I M E NZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase A I M E N A N VZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase A N V L A R Y AZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase L A R Y A S I CZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase I C Q Q N G I VZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase P I V E P E I LZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase R C Q Y V T E KZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase Y V T E K V L AZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase E K V L A A V YZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase V L A A V Y K AZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase L A A V Y K A LZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase A A V Y K A L SZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase H I Y L E G T LZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase Y L E G T L L KZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase Y S H E E I A MZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase E I A M A T V TZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase M A T V T A L RZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase T V T A L R R TZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase V T A L R R T VZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase P A V T G V T FZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase S E E E A S I NZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase E E E A S I N LZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase S I N L N A I NZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase N L N A I N K CZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase W A L T F S Y GZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase A L Q A S A L K
76
Reference Protein P4 P3 P2 P1 P1' P2' P3' P4'Zhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase A S A L K A W GZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase A Q E E Y V K RZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase Q E E Y V K R AZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase A N S L A C Q GZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase S E S L F I S NZhang, Z., C. B. Post, et al. (1996) rabbit muscle aldolase I S N H A YJohnson, R. S. and K. A. Walsh (1994) equine myoglobin D G E W Q Q V LJohnson, R. S. and K. A. Walsh (1994) equine myoglobin Q V K N V W G KJohnson, R. S. and K. A. Walsh (1994) equine myoglobin Q E V L I R L FJohnson, R. S. and K. A. Walsh (1994) equine myoglobin L I R L F T G HJohnson, R. S. and K. A. Walsh (1994) equine myoglobin A E M K A S E DJohnson, R. S. and K. A. Walsh (1994) equine myoglobin T V V L T A L GJohnson, R. S. and K. A. Walsh (1994) equine myoglobin P I K Y L E F IJohnson, R. S. and K. A. Walsh (1994) equine myoglobin Y L E F I S D AJohnson, R. S. and K. A. Walsh (1994) equine myoglobin F I S D A I I HJohnson, R. S. and K. A. Walsh (1994) equine myoglobin I S D A I I H VJohnson, R. S. and K. A. Walsh (1994) equine myoglobin M T K A L E L FJohnson, R. S. and K. A. Walsh (1994) equine myoglobin A L E L F R N D
77
CHAPTER 3
EXPERIMENTAL DETERMINATION OF
PEPSIN SPECIFICITY AND THE EFFECTS OF pH
3.1 Introduction
The second part of this project involved the experimental research, the goal of
which was to increase the data set of pepsin cleavages. The literature search provided a
good starting point, but as stated previously, the amount of data produced was not enough
to accurately determine the trends in pepsin specificity.
Another goal of the experimental research was to determine if the specificity of
pepsin is affected by a change in pH. It is known that pepsin activity is directly affected
by pH (Jones and Landon 2002). Pepsin experiences its highest activity around pH 1.9.
It remains very active down to pH 1 and begins to lose activity around pH 5 (Brier, Maria
et al. 2007).
To address the two main goals of the experimental project various proteins were
digested with pepsin and analyzed using LC/MS/MS. The proteins were digested at three
different pH conditions, pH 1.0, pH 2.5 and pH 4.0. In addition to digesting a selection
of proteins, the bacterium Escherichia coli (E.coli) whole cell lysate was also digested at
the aforementioned pH points and analyzed using LC/MS/MS.
3.2 Instrumentation
Online digestion and sample analysis was performed using UPLC/ESI-MSE. This
method was chosen because it allows for fast and accurate determination of peptides
(Chakraborty, Berger et al. 2007).
78
3.2.1 UPLC and online pepsin digestion
UPLC was chosen for analysis over HPLC because it allows for a faster analysis
time while still obtaining a very high-resolution separation (Swartz 2005). UPLC uses a
smaller particle size (less than 2 μm diameter) which creates very high operating
pressures, up to 15,000 psi. Another plus to using UPLC over HPLC is that UPLC has a
greater sensitivity therefore a smaller sample size can be used (Plumb, Castro-Perez et al.
2004; Swartz 2005).
Online pepsin is digestion preferred over a solution digest for various reasons.
One reason is that online digests allow for greater pepsin efficiency as pepsin becomes
more efficient as the enzyme to protein ratio increases. Compared to a solution digest,
with online digests much more pepsin can be packed into a column, therefore the
concentration of pepsin will be greater, thus increasing efficiency (Wu, Kaveti et al.
2006). The time needed for digestion can also be decreased when performing online
digestions because the efficiency of pepsin is increased. A solution digest that would
take five minutes can be decreased to 20 seconds in a pepsin column (Jones and Landon
2002). One final bonus to performing online digestion is the ability to automate the
process. This was especially important with this project because of the number of
samples that needed to be analyzed.
A schematic of the UPLC setup for online digestion can be found in Figure 3.1.
The sample is injected into valve A. It travels through the loop and is then pushed
through the pepsin column via the auxiliary solvent manager (ASM) which contains a pH
buffer in water. Following digestion the peptides are desalted and concentrated on the
peptide trap. Valve B then switches the trap inline with the binary solvent manager
79
Binary Solvent Manger
Auxiliary Solvent Manager: Pump B
A B
MSC18 Column
A ili S l t M
Pepsin Column
Peptide Trap
Auxiliary Solvent Manager:Pump A
Figure 3.1. Schematic of online pepsin digestion adapted from Wang (2002). The sample is injected into the loop of valve A and is then pushed through the pepsin column with Pump A of the auxiliary solvent manager (ASM) containing H b ff i H O Th ti tid th d lt d d t t d thpH buffer in H2O. The peptic peptides are then desalted and concentrated on the
peptide trap. Valve B then switches to turn the trap inline with the binary solvent manager (BSM). The BSM creates the correct mixture of A and B solvents, where A is 0.05% FA in H2O and B is 0.05% FA in ACN, dependent on the gradient. The sample is then separated on a C18 column with the eluant flowing to the mass spectrometer. Pump B of the ASM delivers the reference material to the mass spectrometer.
80
(BSM). The BSM will then make up the needed mixture of A and B solvents, where A is
0.05% formic acid (FA) in water and B is 0.05%FA in acetonitrile (ACN), according to
the desired gradient. The peptic peptides are then separated on a C18 analytical column.
Following separation the eluant flows into the mass spectrometer for analysis (Wang, Pan
et al. 2002).
3.2.2 Mass spectrometry
For these experiments the technique of MSE was used for mass spectral analyses.
MSE is a data independent method of performing MS and MS/MS analyses. With MSE
both fragment and precursor ion information can be obtained relatively quickly and with
accurate mass in one analysis (Plumb, Johnson et al. 2006).
A diagram of the operation of MSE is displayed in Figure 3.2 (Plumb, Johnson et
al. 2006). The peptides that are separated during the UPLC analysis are introduced into
the mass spectrometer using an electrospray ionization (ESI) source and then enter the
first quadruple where the peptides are separated. The peptic peptides then move into the
collision cell where they are bombarded with either low or elevated energy. After this
step they move into the second quadruple which is operated in V mode (Chakraborty,
Berger et al. 2007).
The collision energy in the gas cell continuously alternates between low and
elevated energies at a specified rate. At the low collision energy a classic MS spectrum is
obtained generating information about the precursor ions. When hit with the elevated
collision energy, information about the fragment ions is obtained. When the elevated
energy is used, the high voltage causes the formation of both the precursor ions and its
81
MS
6
1 2 3 4
5 7
MSE
Figure 3.2. Schematic of the operation of MSE adapted from Plumb (2006). Peptides are separated during the UPLC separation, in this example there are two peptides that co-elute (1), and are introduced into the mass spectrometer via an ESI source (2). From there the peptides are separated in the first quadruple (3) then move into the collision cell (4) then into the second quadruple (5) which is operated in V mode. The collision energy in the gas cell alternates between low and high energy. At low energy precursor ion information is obtained yielding a classic mass spectrum (6). At the elevated energy information is collected for all of the fragment ions (7).
82
fragments. Therefore, throughout one analysis, information is constantly being gathered
about both the precursor and fragment ions. During data processing, the precursor ions
are matched up with their fragment ions based on their common retention times.
(Chakraborty, Berger et al. 2007).
3.3 Materials and Methods
3.3.1 Protein sample analysis
The proteins used for these experiments are commercially available from Sigma-
Aldrich or were obtained in-house. A list of the proteins used for digestion can be found
in Table 3.1. The proteins encompass a wide range of sizes, from 8.6 kDa (Ubiquitin) to
68.0 kDa (Amyloglucosidase).
3.3.1.1 Protein sample preparation
All of the proteins were dissolved into 50 mM TRIS buffer, pH 8.315, to a
concentration of 10 μM. These stock solutions were kept in a -20 °C freezer. 100 μL of
each protein stock solution were diluted into 100 μL of TRIS buffer to a concentration of
5 μM. Abelson tyrosine kinase (Abl), Nef (a small HIV accessory protein) and Ubiquitin
were heated at 60 °C for ten minutes to help reduce the disulfide bonds present. A small
amount of 1 M guanidine-HCl was also added to all samples to aid in digestion. For pH
1.0 analysis the samples were adjusted to pH 1.0 by adding 0.1 M HCl. The samples
were also analyzed at pH 2.5 and 4.0. Fresh aliquots of sample were adjusted to these pH
points by adding a buffer consisting of 50 mM potassium phosphate monobasic and 50
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Table 3.1. Proteins used for digestion
Protein SwissProt ID and Ascension Number
Molecular Weight (kDa)
Number of Residues
Abelson tyrosine kinase (Abl)*
57.4 501
Albumin Albu_Bovin (P02769) 66.0 583 Aldolase Aldoa_Rabit (P00883) 39.5 364
Spectral identification key:Red peaks – y-ionsBl k b iBlue peaks – b-ionsGreen peaks – neutral loss of water and ammonia and immonium ionsGrey peaks – not assigned to y-ions, b-ions, or neutral loss fragments. Other ion
Spectral identification key:Red peaks – y-ionsBlue peaks – b-ionsGreen peaks – neutral loss of water and ammonia and immonium ionsG k t i d t i b i t l l f t Oth iGrey peaks – not assigned to y-ions, b-ions, or neutral loss fragments. Other ion
types and internal fragments are not labeled.
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3.5 Results
Digestion of the nine proteins at pH 1.0 generated 582 peptides (see Table 3.3 at
end of chapter) at 695 cleavage points. pH 2.5 protein digestions produced 482 peptides
(shown in Figures 3.5-3.13 at end of chapter) with 591 different cleavage points and pH
4.0 digestions yielded 235 (see Table 3.4 at end of chapter) peptides and 366 cleavage
points. The E.coli digest at pH 2.5 produced 991 peptides (see Table 3.5 at end of
chapter) at 1368 different cleavage points. There were 49 proteins that were identified in
the E.coli lysate. The sum of data for all digests performed at pH 2.5 is 1473 peptides
and 1959 cleavage points.
While the 49 proteins identified by digesting E.coli lysate seems like a lot, this
number was much lower than expected as E.coli contains thousands of proteins. One
thing that could help to increase the number of proteins and peptides generated is to
further optimize the digestion and chromatography conditions. By increasing the
digestion time and running the analysis with a longer gradient there is a possibility that
more proteins and peptides will be generated.
After the peptic peptide maps were constructed as described in section 3.4 the
data was treated as described in sections 2.3-2.5. A database of cleavages was produced,
followed by data normalization and then the creation of a peptide cleavage map for each
pH. The cleavage data maps along with the corresponding matrix of normalized data
representing the probability of a cleavage occurring between two specific amino acids
can be found in Figures 3.14-3.16 and Tables 3.6-3.8 for pH 1.0, 2.5 and 4.0 respectively.
The peptide cleavage maps show that at all pH points pepsin will often cleave
after most bulky hydrophobic residues such as leucine and phenylalanine. The data also
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Figure 3.14. Cleavage data map pH 1.0
P1
A C D E F G H I K L M N P Q R S T V W YA 1.6 0.9 0.7 0.3 0.9 0.8 0.6 1.7 0.7 1.1 2.7 1.0 0.0 0.8 1.1 1.3 0.7 1.5 1.5 1.5C 0.0 0.0 0.5 2.3 0.0 0.0 0.0 0.0 0.0 1.5 0.0 0.0 1.1 1.8 0.0 0.0 1.0 0.0 0.0 1.5D 0.8 0.0 0.3 1.0 1.7 0.3 0.0 2.0 0.5 0.8 0.0 0.0 0.4 0.0 0.0 0.3 0.5 0.0 0.0 2.3E 1.4 0.6 0.0 1.4 2.0 0.5 2.3 0.5 0.5 1.7 4.5 1.1 0.9 0.6 0.5 1.3 1.2 0.8 2.3 2.1
Albumin 1-21 DTHKSEIAHRFKDLGEEHFKG Albumin 1-24 DTHKSEIAHRFKDLGEEHFKGLVL Albumin 2-24 THKSEIAHRFKDLGEEHFKGLVL Albumin 3-24 HKSEIAHRFKDLGEEHFKGLVL Albumin 21-43 GLVLIAFSQYLQQCPFDEHVKLV Albumin 23-35 VLIAFSQYLQQCP Albumin 25-38 IAFSQYLQQCPFDE Albumin 25-42 IAFSQYLQQCPFDEHVKL Albumin 25-45 IAFSQYLQQCPFDEHVKLVNE Albumin 27-38 FSQYLQQCPFDE Albumin 30-45 YLQQCPFDEHVKLVNE Albumin 31-45 LQQCPFDEHVKLVNE Albumin 31-77 LQQCPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKV Albumin 34-81 CPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLR Albumin 59-82 HAGCEKSLHTLFGDELCKVASLRE Albumin 107-121 DDSPDLPKLKPDPNT Albumin 112-133 LPKLKPDPNTLCDEFKADEKKF Albumin 113-133 PKLKPDPNTLCDEFKADEKKF Albumin 125-142 EFKADEKKFWGKYLYEIA Albumin 126-144 FKADEKKFWGKYLYEIARR Albumin 138-154 LYEIARRHPYFYAPELL Albumin 141-153 IARRHPYFYAPEL Albumin 141-154 IARRHPYFYAPELL Albumin 141-155 IARRHPYFYAPELLY Albumin 145-156 HPYFYAPELLYY Albumin 154-164 LYYANKYNGVF Albumin 169-191 QAEDKGACLLPKIETMREKVLAS Albumin 170-188 AEDKGACLLPKIETMREKV Albumin 177-191 LLPKIETMREKVLAS Albumin 177-208 LLPKIETMREKVLASSARQRLRCASIQKFGER Albumin 199-211 CASIQKFGERALK Albumin 212-227 AWSVARLSQKFPKAEF Albumin 213-227 WSVARLSQKFPKAEF Albumin 275-293 KECCDKPLLEKSHCIAEVE Albumin 276-290 ECCDKPLLEKSHCIA Albumin 277-300 CCDKPLLEKSHCIAEVEKDAIPEN Albumin 279-319 DKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQ Albumin 292-303 VEKDAIPENLPP Albumin 328-338 SFLYEYSRRHP Albumin 331-345 YEYSRRHPEYAVSVL Albumin 334-354 SRRHPEYAVSVLLRLAKEYEA Albumin 337-357 HPEYAVSVLLRLAKEYEATLE Albumin 355-399 TLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKLGE Albumin 369-390 YSTVFDKLKHLVDEPQNLIKQN Albumin 402-422 FQNALIVRYTRKVPQVSTPTL Albumin 406-422 LIVRYTRKVPQVSTPTL Albumin 407-419 IVRYTRKVPQVST Albumin 407-421 IVRYTRKVPQVSTPT Albumin 407-422 IVRYTRKVPQVSTPTL Albumin 408-421 VRYTRKVPQVSTPT Albumin 418-441 STPTLVEVSRSLGKVGTRCCTKPE Albumin 419-432 TPTLVEVSRSLGKV Albumin 419-440 TPTLVEVSRSLGKVGTRCCTKP Albumin 443-463 ERMPCTEDYLSLILNRLCVLH Albumin 448-466 TEDYLSLILNRLCVLHEKT Albumin 460-470 CVLHEKTPVSE Albumin 460-511 CVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHAD Albumin 464-482 EKTPVSEKVTKCCTESLVN Albumin 490-505 LTPDETYVPKAFDEKL Albumin 496-515 YVPKAFDEKLFTFHADICTL Albumin 529-543 VELLKHKPKATEEQL Albumin 529-545 VELLKHKPKATEEQLKT Albumin 529-547 VELLKHKPKATEEQLKTVM
Albumin 1-21 DTHKSEIAHRFKDLGEEHFKG Albumin 28-58 SQYLQQCPFDEHVKLVNELTEFAKTCVADES Albumin 32-79 QQCPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVAS Albumin 71-78 GDELCKVA Albumin 77-105 VASLRETYGDMADCCEKQEPERNECFLSH Albumin 103-137 LSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKY Albumin 113-133 PKLKPDPNTLCDEFKADEKKF Albumin 130-159 EKKFWGKYLYEIARRHPYFYAPELLYYANK Albumin 146-158 PYFYAPELLYYAN Albumin 154-164 LYYANKYNGVF Albumin 179-192 PKIETMREKVLASS Albumin 189-202 LASSARQRLRCASI Albumin 231-262 TKLVTDLTKVHKECCHGDLLECADDRADLAKY Albumin 235-271 TDLTKVHKECCHGDLLECADDRADLAKYICDNQDTIS Albumin 240-277 VHKECCHGDLLECADDRADLAKYICDNQDTISSKLKEC Albumin 241-277 HKECCHGDLLECADDRADLAKYICDNQDTISSKLKEC Albumin 253-271 ADDRADLAKYICDNQDTIS Albumin 261-289 KYICDNQDTISSKLKECCDKPLLEKSHCI Albumin 269-290 TISSKLKECCDKPLLEKSHCIA Albumin 278-287 CDKPLLEKSH Albumin 287-327 HCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLG Albumin 291-306 EVEKDAIPENLPPLTA Albumin 292-306 VEKDAIPENLPPLTA Albumin 292-307 VEKDAIPENLPPLTAD Albumin 292-308 VEKDAIPENLPPLTADF Albumin 308-323 FAEDKDVCKNYQEAKD Albumin 373-386 FDKLKHLVDEPQNL Albumin 374-386 DKLKHLVDEPQNL Albumin 378-390 HLVDEPQNLIKQN Albumin 381-396 DEPQNLIKQNCDQFEK Albumin 384-399 QNLIKQNCDQFEKLGE Albumin 395-406 EKLGEYGFQNAL Albumin 407-419 IVRYTRKVPQVST Albumin 466-483 TPVSEKVTKCCTESLVNR Albumin 485-512 PCFSALTPDETYVPKAFDEKLFTFHADI Albumin 488-501 SALTPDETYVPKAF Albumin 493-528 DETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTAL Albumin 496-514 YVPKAFDEKLFTFHADICT Albumin 506-521 FTFHADICTLPDTEKQ Albumin 516-528 PDTEKQIKKQTAL Albumin 529-542 VELLKHKPKATEEQ Albumin 542-555 QLKTVMENFVAFVD