Chapter one: Recent development in protein affinity …...When developing a FAC method, the handbook of affinity chromatography by Hage et al[8], and the paper on practical protocol

13

Chapter one:

Recent development in protein affinity selection mass

spectrometry

Chapter one

14

Abstract

This review intends to provide an overview of protein affinity selection methodologies

that can be coupled to mass spectrometry. It will shortly address the principle of each of

the methodologies, but will mainly focus on the developments in recent years, and the

applicability of the methodologies to lead generation or other research fields important in

drug discovery.

Introduction

The development of new lead compounds in drug discovery has been of

continuous importance over the past decades. However, high attrition rates and the

decreasing number of new drug approvals in the past years have increased the necessity

for new development tools. Protein affinity selection methods utilizing mass

spectrometry are amongst the more recently developed methodologies that can be a

valuable addition to traditional drug discovery techniques. They distinguish themselves

by utilizing the very high sensitivity and selectivity that is inherent to mass spectrometric

detection, while retaining the biological specificity that is typical for commonly used

plate reader assays.

The use of mass spectrometry has a number of consequences for the bio-assay. A

positive implication is the fact that no labels are required in mass spectrometry. This

widens the application area for these methods, including target proteins for which no

label is available or can be developed. A second advantage is that the mass spectrometer

measures the actual compound that has the affinity for the protein, instead of a labeled

competitor. Since protein selection methodologies deliver simultaneous biological and

structural data on the compound to be analyzed, the detection of false positives due to

library impurities or degradation of the compounds in the mixture is very improbable.

Several methodologies have been developed using direct measurement of a

protein-ligand complex in mass spectrometry, and a number of interesting reviews have

been published recently.[1-3] However, many of these methods require extensive

Chapter one

15

optimization in order to combine protein-ligand complex stability with conditions

suitable for mass spectrometric detection. Secondly, advanced mass spectrometric

method development is required to distinguish the protein-ligand complex from the

unbound protein without prior separation. As a result, these methodologies have not yet

shown to be universally applicable to a large number of target proteins. However, they do

provide the most direct evidence of protein-ligand complexation, provided the

assumption that gas-phase complexation in the mass spectrometer can be used as a model

for in vivo complexation.[4] Furthermore, many protein-ligand binding assays do not use

mass spectrometry. An excellent review on this topic, published by de Jong et al., focuses

on assay technology used in high throughput screening, with a strong emphasis on

photometric detection.[5]

In this paper a comprehensive overview of recent developments in the field of

protein affinity selection methods that utilize mass spectrometry is provided. The scope is

limited to methodologies that involve a step that separates bound and unbound protein

before detection. All these methodologies consist of the same four steps: (1)

Complexation of the protein and test ligand. (2) Separation of non-bound compounds

from the protein-ligand mixture. (3) Elution/dissociation, resulting in the release of the

free, bioactive ligand, and (4) Detection of the eluted ligand by mass spectrometry. An

overview is provided of the various affinity selection methodologies that are being

applied in modern-day drug discovery, such as size-exclusion chromatography, (pulsed)

ultrafiltration, surface plasmon resonance and immobilized or dynamic protein affinity

selection methodologies.

Affinity Chromatography

Affinity chromatography is a process in which a compound, be it a protein, small

molecule, antigen, antibody or any such biologically active agent is immobilized on a

solid support. When a mixture of analytes or a complex sample containing an analyte is

injected onto the column containing this solid support, the analyte that shows affinity for

the immobilized compound gets retained, and separated from the rest of the sample. The

Chapter one

16

method originates from the late sixties, when it was used to extract and purify

enzymes[6] or antibodies[7]. These methodologies were based upon solid supports

consisting of mainly sepharose or agarose. These materials were known for their low

non-specific adsorptive properties, and their relative ease of modification, allowing easy

immobilization of the desired target. However, when the methodology developed towards

affinity chromatography, with the aim of not only extracting and purifying the analytes,

but also ranking them according to their affinity, it became a necessity to use support

materials that were better equipped for the flow rates and capillary pressure that is

common in HPLC. The use of pressure resistant solid supports is often referred to as the

defining advantage of high performance affinity chromatography over conventional

affinity chromatography, and was a starting point for a rapid expansion of the number of

applications published in this field.[8]

Many modern-day innovations are aimed at improving the immobilization of the

target on the backbone and improving the quality of the materials used in the solid

support. However, most of them use two approaches for the actual proof of principle. The

first is frontal affinity chromatography, in which a known concentration of analyte is

infused onto an immobilized protein column, and affinity is calculated based on the

saturation time and the shape of the breakthrough curve. The second is zonal elution, in

which a small amount of analyte is injected onto an immobilized protein column together

with a known amount of a competitor. By varying the concentration of the competitor

and measuring the retention time of the analyte, its affinity can be calculated.

Frontal affinity chromatography

The concept of frontal affinity chromatography is more complicated than it

appears at first sight. A target, very often a protein or receptor, is covalently immobilized

on a column. An analyte is then infused onto the column at a known concentration, and

the analyte concentration exiting the column is measured. In the beginning this amount

will be very low, because there is a large amount of free receptor for the analyte to bind

to. However, as a larger fraction of the immobilized receptor is bound, the amount of

analyte being measured will slowly increase. At some point, all the receptor on the

Chapter one

17

column will be bound to the analyte, and the signal in the detector will equal that of the

concentration of analyte that is being infused. However, the speed at which this happens

is dependant on the association (Ka) and dissociation constants (Kd) of the protein-analyte

interaction. An analyte with fast dissociation kinetics will have a longer saturation time

than one with slow dissociation, because during the process some of the protein-analyte

complexes will dissociate, increasing the fraction of unbound protein. As a result the

affinity of the analyte for the protein can be calculated by varying the concentration of

analyte, and determining the absolute amount of analyte needed to reach 50% saturation

for every concentration. A typical result for such an experiment is shown in figure 1.1[9].

Figure 1.1: A frontal analysis experiment performed with L-tryptophan on an immobilized human

serum albumin column. The concentration of applied L-tryptophan (from left to right)

was 100, 50, 25 and 12.5 µM. The flowrate was 0.25 mL/min and t0 was 3.6 min.

Reproduced from ref [9].

When developing a FAC method, the handbook of affinity chromatography by

Hage et al[8], and the paper on practical protocol development by Ng et al.[10] should

provide all the theoretical and practical background required. This review will focus on

the most significant developments in FAC methodologies that employ mass spectrometric

detection. These developments are currently focused on two fundamental assay

properties: the improvement of the solid support material to facilitate easier protein

Chapter one

18

immobilization, reduce non-specific binding and stabilize certain classes of target

proteins, and the miniaturization of the protein affinity column, in order to minimize

protein consumption. Prime examples are the use of artificial immobilized membranes

(AIM), monolithic affinity columns, and miniaturization of the affinity columns. Next to

these fundamental improvements, a significant number of papers have been published

expanding the number and types of targets successfully immobilized.

The most significant development in the field of novel solid supports has been the

application of immobilized artificial membranes (IAM) to form immobilized receptor

columns. Ever since the introduction of FAC in conventional HPLC systems, the target

proteins were immobilized mainly on silica, glass or polystyrene material. One of the

largest disadvantages of these solid support materials was the fact that many proteins lost

a major part of their activity upon immobilization. In order to widen the applicability of

FAC, a solid support contributing to protein stability was required. IAMs were developed

by Pidgeon et al. in 1989 [11], consisting of silica beads covered with membrane lipids to

be used as a stationary phase in chromatography. Many receptor types have been

immobilized on IAMs since then, and in recent years Beigi et al. have shown the

immobilization and screening of analytes to G-protein coupled receptors.[12,13].

Recently, Temporini et al. have successfully immobilized the orphan receptor GPR17 on

artificial immobilized membranes, including an affinity ranking experiment, the results of

which are shown in figure 1.2.[14] Also, Moaddel et al. published a comprehensive guide

on IAM development and preparation, allowing fast implementation by groups with little

experience in the field.[15]

The other two major developments are often applied in conjunction. Because

columns on microfluidic chips or in nano-LC scale experiments are very small, they have

very low protein consumption. Furthermore, because these small columns are hard to

pack with conventional bead type materials, they have been a prominent target for the

development of monolithic affinity columns. A very elegant method requiring complex

implementation was published by Besanger et al. immobilizing membrane-bound

nicotinic acetylcholine receptor in a monolithic column built up from diglycerylsilane

polymers.[16] By adding the right amount of polyethylene glycol, the membrane and the

active receptor were wholly incorporated in a silica monolith, and successfully used in a

Chapter one

19

FAC-MS experiment comparing the affinity of epibatidine to a benchmark method.

However, slight adjustments in the manufacturing procedure could lead to major changes

in the monolith formation and the receptor activity. Okanda et al. published a monolithic

silica column with immobilized lectins incorporated with suitable properties for affinity

ranking in both nano-LC and capillary electrophoresis.[17] Finally Feng et al. reported

the fabrication of a monolithic silica column with incorporated Fe3+

, allowing nano-scale

immobilized metal affinity chromatography (IMAC).[18]

Figure 1.2: Affinity ranking of 3 ligands to GPR17 receptor immobilized on an IAM using frontal

affinity chromatography-mass spectrometry. From top to bottom UDP (Kd = 1140 nM),

MRS2197 (Kd = 508 nM) and cangrelor (Kd = 0.7 nM). Reproduced from ref [14].

Zonal Elution

Zonal elution is the most straightforward method of performing affinity

chromatography. Basically it is a classic competition experiment, implemented in a

HPLC system. A known and constant amount of the analyte is injected in a small amount

onto the affinity column, in the addition of a competitor. When a series of experiments is

performed using a varying concentration of competitor, the retention of the analyte will

vary. For a high concentration of competitor, analyte retention will be low, because a

Chapter one

20

large fraction of the available protein bound to the column is occupied by the competitor.

Likewise, for a low concentration of competitor, analyte retention will be high. The

advantage of this method over FAC is the small amount of analyte consumption, which is

useful when analytes are scarce, expensive or hard to synthesize. However, frontal

analysis provides more information per experiment, allowing for instance the

determination of the number of binding sites per column and the association constants of

the protein-analyte complexes. Because of the limited amount of data collected, most

affinity chromatography development in pharmaceutical industry is aimed at frontal

analysis. Zonal elution is nowadays more often used as a means to acquire additional data

on an interaction observed in frontal analysis, such as the location of a binding

site.[19,20] Furthermore, many of these methodologies involve other means of detection

than mass spectrometry. One of the few recently published method based solely on zonal

elution has been developed by Bertucci et al., who investigated the binding dynamics of

HIV inhibitors to human serum albumin using zonal elution.[21].

In conclusion, it is apparent that frontal affinity chromatography is a prominent

technique in pharmaceutical research, because of the high amount of data produced, and

the wide variety of targets that can be immobilized. In recent years, FAC applicability has

extended to include G-protein coupled receptors, a hot target in pharmaceutical research

and notoriously hard to stabilize when used in bio-assays. Due to the fast development of

frontal analysis, zonal elution is used more as an auxiliary technique. When comparing

frontal analysis to other affinity selection techniques, two drawbacks of the technology

must be addressed. First, for every target that is immobilized, extended controls must be

performed to check whether the immobilized protein has the same functionality as the

protein in native conditions. Immobilization influences the freedom of movement of the

protein, and might impede binding to certain binding sites, thus decreasing the affinity of

certain ligands to the protein, causing false negatives. Secondly, immobilized protein

columns have a very limited stability. If an HTS screening assay would be developed

using FAC, it would require a large number of functionally equal columns to be

produced, which might be challenging.

Chapter one

21

Affinity Selection

Affinity selection, also known as affinity capture or affinity trapping

methodologies, are similar to affinity chromatography in the sense that their mechanism

of action depends on possible analytes binding to immobilized proteins on a solid

support. However, in affinity selection no chromatographic separation of ligands is

involved. The conditions in which the binding occurs are optimized to stabilize the

protein-analyte complex, wash off non-binders, and then dissociate the complex to detect

the binding analytes. In order to identify binders, most of these methodologies employ

mass spectrometric detection. Because the compounds are not separated based on their

affinity for the receptor, the resulting analytes can not be ranked, but will only be

grouped in binders and non-binders. Optimizing the threshold to distinguish these two

groups, in order to eliminate false negatives, and limit false positives, is one of the most

challenging aspects of affinity selection development.

Although affinity selection in one form or another has been around for decades

[22-24], it has become more prominent in bio-analysis upon the introduction of the lectin-

based affinity materials, used to extract bacteria and carbohydrates with a specific

binding motif for MALDI-TOF analysis.[25,26]. An example of results obtained using

these lectin-based affinity capture surfaces is shown in figure 1.3. These methodologies

allowed extremely sensitive detection of target proteins that were hard to analyze in any

conventional way due to the complex matrices they were in. Since that time a number of

very specific affinity materials have been developed and employed with considerable

success. Krugman et al. presented their work in which a novel family of proteins found in

leukocytes bound specifically to phosphoinositide affinity material, and could be isolated

and identified for the first time.[27] Kong at al. described a diamond based affinity

material with the property of extracting any kind of protein from any complex mixture.

It’s lack of specificity was countered by a direct coupling to a MALDI-TOF, allowing

mass spectrometric identification.[28] Recently, Ferrance has shown the feasibility of

immobilizing proteins on gellan beads (a polysaccharide).[29] The optical transparence

of these beads allows direct measurement of the amount of protein successfully

Chapter one

22

immobilized on the material. Freije et al. developed a sepharose material with a small

peptide immobilized on it in order to select metalloproteases based on their activity [30],

and successfully implemented this material as a sample enrichment step in an online

affinity selection-trypsin digestion-MS proteomics approach able to identify the proteases

and rank their activity [31]. Finally, Johnson et al. utilized affinity selection by

immobilizing an in-house developed capture ligand to selectively extract protein A from

Staphylococcus Aureus, resulting in an easy to optimize affinity material synthesis

procedure that can be used on a wide variety of target proteins.[32]

Figure 1.3: MALDI-TOF mass spectra resulting after the extraction of Sinbis virus (MW = 51800)

from cell culture using (a) lectin-modified affinity capture surface, and (b) unmodified

affinity membrane. BSA is bovine serum albumin. Reproduced from ref [25].

Chapter one

23

Immobilized Metal Affinity Chromatography (IMAC) comprises a large number of

methods similar to protein affinity chromatography, but with one difference putting it

beyond the scope of this paper. It separates proteins based on their affinity to an

immobilized metal ion on a solid support. Instead of protein ligand affinity, metal-ligand

affinity is determined. This separation mainly depends on the number and location of

histidine residues in the proteins to be analyzed, and to a lesser extent cysteine and

tryptophane residues.[33] However, when a protein is His-tagged, a chain of six

histidines is artificially implanted into the protein, resulting in a very high affinity

between the His-tag and the immobilized metal ion. Because of this high affinity, there is

no longer any chromatographic separation involved, the IMAC column simply traps any

His-tagged protein from any matrix. As a consequence, nowadays this is the most widely

used method of protein purification. A perfect recent example of which is shown by

Cheek et al. who developed a monolithic IMAC column in order to purify His-tagged

lentiviral vectors.[34] Jonker et al. developed a dynamic protein affinity selection

methodology utilizing immobilized metal ions, by trapping an in-solution formed protein-

ligand complex on a small nickel-loaded column.[35] This allows complex formation

under native conditions, using the immobilized metal column merely to separate the

bound and unbound fraction, followed by mass spectrometric detection.

Another variant of affinity capture methodologies that has been increasingly

popular is ligand fishing using magnetic particles. A target protein is immobilized on

magnetic beads, and added to an oftentimes complex mixture of possible ligands. All

possible ligands are allowed to bind to the immobilized protein, and the mixture is

exposed to a magnetic field, retaining the beads and anything bound to them, while the

unbound fraction is removed. The regular use of magnetic beads for ligand fishing

originates in the early nineties, when streptavidin-coated beads were first used to purify

biotin-labeled proteins[36], and antigens immobilized on magnetic beads were applied

for antibody purification.[37]. Within a couple of years this lead to two major

developments: immobilized metal magnetic beads, able to trap His-tagged proteins[38],

and the paper by Mehta et al. first describing direct immobilization of proteins on

magnetic beads.[39] Nowadays, most of these types of magnetic particles are being

produced for the mass-market, and are available to anyone. This has lead to a rapid

Chapter one

24

increase in the number of papers published utilizing magnetic beads. One example of a

recent development of the use of immobilized metal magnetic particles is magnetic bead

dynamic protein-affinity selection, in which the beads are used to trap a His-tagged

protein-ligand complex from a sample, allowing protein affinity selection in much more

complex mixtures, the results of which are shown in figure 1.4.[40]

Figure 1.4: Weak binder identification using magnetic bead dynamic affinity selection as sample pre-

treatment for a mixture of possible ligands. Norethisteron is successfully extracted as the

only binder in the set of compounds. Reproduced from ref [40]

Furthermore, Meyer et al. developed a gradient magnetic fishing methodology utilizing a

non-linear magnetic field to capture superoxide dismutase from crude whey.[41] In the

field of protein immobilization on magnetic particles the work of Marsza et al is a prime

example of the vast body of work published by the group of Wainer et al., employing

Chapter one

25

magnetic beads coated with heat shock protein, and effectively using them for ligand

fishing.[42] Finally, Hu et al. published an enzyme inhibition screening assay utilizing

enzymes immobilized on magnetic beads with considerable success.[43]

In conclusion, affinity selection methodologies are rapidly developing, and are

important tools for future drug discovery. Their advantage is the wide applicability of

affinity selection, the easy compensation that can be made for non-specific binding, and

their relatively high sensitivity. The major drawback is the fact that affinity ranking using

these methodologies is not possible. The results of these methods will be a group of

binders and a group of non-binders. The biggest challenge is to eliminate false negatives,

and limit false positives as much as possible. In order to rank the affinities of the group of

binders, most of the other methods in this paper are more suitable.

Ultrafiltration

Traditional

In the early eighties, ultrafiltration was developed as a means to measure protein-

ligand interactions in solution based complexes.[44,45] The concept of the technique is

rather straightforward. A certain amount of pressure is applied to an amount of liquid

containing protein-ligand complexes as well as free ligand and protein. This can be

achieved by applying pressure using a pump, vacuum or centrifugal force. The

ultrafiltration unit contains a molecular weight cut-off membrane, allowing solvents and

non-bound small molecules to pass through, but retaining proteins and protein-ligand

complexes. This achieves effective separation of the bound and non-bound fractions of

analyte, allowing detection of the bound ligand by any available detection method. The

large advantage of this method over other existing methodologies is the fact that complex

formation takes place in solution, allowing the protein the same degree of freedom it

would in vivo. Most competing methods at that time used immobilized proteins, with the

drawback that immobilization could influence the binding properties of the protein.

Chapter one

26

Since that time, many interesting developments have followed. Wieboldt et al.

developed an online ultrafiltration MS methodology to identify affinity for an antibody in

a testset of structurally very similar compounds (figure 1.5).[46]

Figure 1.5: Typical results of a immunoaffinity ultrafiltration methodology. The testset consists of

20-30 benzodiazepines. In (a) anti-nitrazepam is used as the affinity ligand before

ultrafiltration. In (b) it is anti-flunitrazepam. (c) is the LC-MS reference chromatogram of

the complete testset. Reproduced from ref [46].

Weisiger et al. developed a methodology in which the complex in vivo interaction

between bilirubin and albumin is studied utilizing serial ultrafiltration.[47] In order to

study this interaction, a series of successive ultrafiltration steps is required in order to

remove impurities in the sample. This resulted in very accurate affinity constant, allowing

the study of factors such as albumin concentration and pH, which have relatively small

influence on the affinity constant. A number of groups have developed methodologies

using continuous ultrafiltration.[48,49] In this technique, a fixed amount of protein is

injected into an ultrafiltration chamber. The analyte is then pumped through the chamber.

Much like frontal affinity chromatography, at first the analyte will bind to the protein

available in the ultrafiltration unit, but upon binding the amount of available binding sites

Chapter one

27

will decrease, and an increasing amount of analyte will pass through the molecular

weight cut-off membrane and be detected. A recent example of these methods using

automated continuous ultrafiltration is published by Heinze et al.[50], utilizing it to

determine antibody-protein binding in human plasma. Fung et al. were one of the first to

automate ultrafiltration for high throughput analysis for Schering-Plough.[51] Some other

recent publications include the work of Comess et al, who developed a high throughput

serial ultrafiltration methodology to screen a library of compounds for affinity to a

pharmacologically relevant streptococcal enzyme.[52] Finally, Li et al developed an

online coupled ultrafiltration LC-MS methodology that was used to screen natural

extracts for �-glucosidase inhibitors.[53]

Pulsed Ultrafiltration

The development of pulsed ultrafiltration by van Breemen et al. significantly

increased the applicability of ultrafiltration.[54] Instead of filtration of an entire sample, a

small amount of sample was injected into the ultrafiltration unit. The liquid flow would

push the non-bound fraction through the molecular weight cut-off membrane, to waste.

Afterwards the bound ligand is dissociated from the protein by buffer adjustment. The

bound fraction is then pushed through the membrane, and immediately measured and

identified by mass spectrometry. The two main advantages over traditional ultrafiltration

are that: (i) less analyte was needed; (ii) receptors could be reused if a non-destructive

dissociation buffer was applied. The methodology could be applied to complex mixtures

as well as combinatorial libraries, and it was functioning online, automated and suitable

for high throughput screening. Since the publication of this methodology, most novel

ultrafiltration applications have used some or all of the characteristics of pulsed

ultrafiltration, very often without identifying them as such. As a result, there no longer

exists a strict division between traditional and pulsed ultrafiltration. The technology and

terminology involved are very often used incorrectly. However, a significant number of

interesting advancements has been published since.

Some successful applications of pulsed ultrafiltration include the work of Shin,

Liu and Cheng, all in the group of van Breemen, who used pulsed ultrafiltration to study

metabolic stability[55], inhibitors of protein aggregation[56] and ligands for human

Chapter one

28

retinoid X receptor alpha[57]. In the field of ultrafiltration, most development is focused

on new applications, and not on improving the technology.

When reviewing ultrafiltration, it is apparent that the two biggest advantages of

ultrafiltration based affinity selection methodologies are (i) in solution complex

formation, and (ii) the possibility to reuse the receptor. However, in practice the most

common problems occurring when implementing ultrafiltration are leakages, protein

denaturation and consequentially reduced activity of the receptor mixture and non-

specific binding to the ultrafiltration membrane. As a consequence, the use of competing

technologies is growing faster than the use of ultrafiltration.

Size exclusion Chromatography

The concept of separating compounds by size has been developed around 1964, in

the form of gel permeation chromatography, mainly applied to the analysis of high

molecular weight polymers.[58] The concept is as simple as elegant. A cross-linked

dextran gel is used as column material. The porosity of the gel will determine its

separation properties, resulting in less retention for larger molecules, and more retention

for smaller molecules. This is caused by the diffusion of the smaller molecules into pores

in the polymeric gel. Because small molecules can enter these pores and the larger

(proteins) can not, their retention will be longer than that of the protein. With the

development of more advances gels and other size exclusion materials, the separation

power of size exclusion-based methods has steadily increased since their inception.

Mainly due to favorable results obtained in drug discovery projects performed by the

major pharmaceutical corporations, size exclusion affinity selection coupled to mass

spectrometry is one of the most important protein-ligand interaction screening

methodologies in current pharmaceutical research. Size exclusion methods are easy to

automate and very suitable for implementation in High Throughput Screening (HTS)

assays. As a result, they are the fastest methods of all discussed in this paper.

Most gel permeation chromatography has been developed in order to function in

spin columns, in which the sample is pipetted onto the gel, but will not permeate the gel

Chapter one

29

unless exposed to centrifugal force.[59,60] The most direct successor to the classic gel

permeation chromatography is SpeedScreen technology developed by Novartis.[61] It

consists of a double 96-well plate format. The upper contains size exclusion gel, and has

holes in the bottom of the plate. An in-solution protein-ligand incubation mixture is

pipetted on top of the gel, and it is placed upon the second plate, a collection plate. The

incubate is separated due to centrifugal force, and bound and unbound ligands are

separated (figure 1.6). The 96-well plate is then analyzed using a standard LC-MS

system. The system screens one 96-well plate within 10 minutes, has routinely been

applied by Novartis and resulted in a number of lead compounds.[62] However, the

preparation of the 96-well plates is an intricate process, and might complicate its

application outside the laboratory where it was developed. Since its development, no new

developments have been published on the subject by Novartis or any of their competitors.

Figure 1.6: Results obtained using the Speedscreen methodology. Olomoucine and Staurosporine,

both known ligands for Protein Kinase A are isolated and detected from a mixture of 400

non-binding compounds. Reproduced from ref [61].

Chapter one

30

Based on the same concept, size exclusion columns are being used in almost all

branches of protein analysis. A much larger variety of materials is available in size

exclusion chromatography than in gel permeation chromatography (which traditionally

uses Sephadex gel), and the reusability of these materials allow the use of these columns

in online LC methods. In 2004, Neogenesis developed the first automated HTS assay

using online size exclusion chromatography coupled to mass spectrometry for the

assessment of protein-ligand interactions.[63] This method, named Automated Ligand

Identification System (ALIS), uses a standardized SEC-LC-MS setup, but contains an in-

house developed resin as the size exclusion material. The results obtained by ALIS have

been very impressive and next to fast affinity screening (figure 1.7, [64]) include the

recent discovery of a lipid phosphatase inhibitor involved in Diabetes type 2[65] and an

acetyl coenzyme A carboxylase ligand that is involved in antibiotics development[66].

Furthermore, it appears to be the only HTS methodology able to screen for ligands of

membrane proteins, such as G protein coupled receptors (GPCRs), which are prime

candidates to be the target of the next generation of drugs.[67]

Following the success of ALIS, a number of groups have developed innovative

applications for size exclusion affinity measurements, including some other

pharmaceutical companies. Flarakos et al. have developed a methodology able to assess

and rank ligand binding towards human serum albumin based on automated size

exclusion chromatography coupled online to a two dimensional LC-MS system, allowing

the screening of ligands with protein affinities over 3 orders of magnitude. [68] Schmidt

et al. developed a SEC-LC-MS method to analyze intact protein-arsenic complexes.[69]

Wolf et al. published a variant of SEC-LC-MS utilizing ICP-MS, especially optimized to

measure metal-ions, in this case urinary Cadmium bound to metallothionein.[70] Adam et

al. have recently expanded their earlier work in order to develop a SEC-LC-MS

methodology based on conformational changes in the protein upon binding to one of two

parnafungin isomers, thereby showing the specificity that can be accomplished using

SEC.[71]

Chapter one

31

Figure 1.7: Binding affinities calculated from ALIS data. Dissociation constants are calculated by

performing saturation experiments for 4 ligands towards muscarinic M2 acetylcholine

receptor. Affinity constants are calculated by performing displacement competition

binding experiments using the known strong binder N-methylscopolamine. Reproduced

from ref [64].

Concluding, size exclusion chromatography coupled to mass spectrometry is a very

powerful methodology, with a wide applicability, easy automation and proven results in

both fundamental research and high throughput screening. Its only disadvantages are non-

specific binding to the size-exclusion material, which can result in false positives or

negatives, and the fact that reusability of the columns is significantly reduced when more

complex protein matrices are researched.

Affinity capillary electrophoresis

Capillary electrophoresis has been used for the separation of proteins and peptides

for several decades, however, around 1992 several groups simultaneously developed

methodologies to assess protein-ligand interactions[72], or protein-DNA binding[73],

using capillary zone electrophoresis. Since that moment, the field of affinity capillary

Chapter one

32

electrophoresis (ACE) has gradually expanded and increased its applicability.[74,75]

Two major technological breakthroughs have contributed to the development of modern-

day ACE: coupling to mass spectrometry and the use of microfluidic chip technology.

Novel methodologies comprising either or both of these elements have resulted in

detection limits comparable to HPLC methodologies and proven their worth in

pharmaceutical development. When focusing on protein drug interactions, ACE

methodologies need to be separated into two categories: mobility shift methodologies and

immobilized protein methodologies.

Mobility shift methodologies

In mobility shift methodologies, the protein and ligand form a non-covalent

complex in solution. For compounds with a very slow koff, a small amount of this

incubation mixture is injected into a standard CE-MS system, and the constituents of the

mixture are separated based on the fact that the non-covalent protein ligand complex has

a distinctly different electrophoretic mobility than the non-bound protein and ligand. The

concentration of the bound and non-bound fraction can be quantified using mass

spectrometry, and protein ligand affinity can be calculated from these results. An

interesting example is published by Groesll et al. coupling CE to ICP-MS. This enabled

the researchers to assess binding between Gallium (III) based anti-cancer drugs and

serum proteins.

In the case of protein-ligand complexes with a faster koff, the complex will

dissociate quickly upon injection into the CE-MS. A small adjustment is made to

facilitate these experiments. In these cases, either the ligand or the protein will be added

to the electrophoresis buffer, thus changing the mobility of its counterpart in the

capillary. A Lineweaver-Burke plot is necessary to extrapolate the affinities from these

kinds of experiments. Many varieties of mobility shift methodologies have been

developed for various applications. The most frequently used is frontal analysis capillary

electrophoresis (FACE), distinctly different from frontal analysis chromatography in the

fact that the protein is not immobilized on a solid support, but incubated with a ligand

before injection. Recently Fermas et al. developed a methodology aimed at the study of

complexation between antithrombin and heparin pentasaccharide, a process that is

Chapter one

33

important for many biological functions.[76] They combined a continuous infusion of a

pre-incubated protein-ligand complex solution with a mass spectrometric detection. This

resulted in dramatically improved limits of detection when compared to competing MS

methodologies, allowing speculation on the future development of ligand fishing

methodologies based on FACE.

Furthermore, since the development of FACE, it has become apparent the

methodology can be used to determine protein-ligand affinity for a great variety of targets

and libraries. A small number of examples published in 2009: The interaction between

Lidocaine and hyaluronic acid[77], the interaction between a large number of drugs and

the human liposome[78] and affinity studies performed on a large testset of drugs and

human serum albumin[79]. It can safely be concluded that FACE is being routinely

employed in bioanalytical research. However, a number of improvements are still in

development, especially aimed at reducing the amount of analyte consumption. Partial

filling techniques are used to save on analyte consumption and have been around for a

little more than a decade, allowing the analysis of very expensive or complicated to

synthesize analytes.[80-82] However, these methods have a significantly higher limit of

detection than frontal analysis methodologies. A recent development to counter this

problem is partial filling multiple injection affinity capillary electrophoresis. Multiple

injections allow an electrophoretic pre-concentration in the capillary, while still using

significantly less analyte than FACE. In 2007, Zavaleta et al. developed a partial filling

multiple injection ACE methodology to study the binding of various antibiotics to D-Ala-

D-Ala-terminus peptides, thus establishing the applicability and efficiency of the

methodology.[83] More recently, Almeda et al. used a variation on this methodology to

detect antibody-antigen complexes, an application for which relatively low limits of

detection are required.[84]

Immobilized protein methodologies

Immobilized protein methodologies involve any method in which the target

protein is immobilized on a solid support, be it silica, (magnetic) beads, microfluidic

chips or any other. When a sample containing one or more possible ligands to the target

protein is injected into the system, it will bind to the immobilized target protein, thus

Chapter one

34

facilitating sample clean-up, pre-concentration, or performing a buffer change before

detection. In the past couple of years, these methodologies have especially increased in

relevance due to the relative ease of immobilizing protein on microfluidic chips and

magnetic beads. Yang et al. published an immunoaffinity CE method incorporating an

anti-�FP (�-fetoprotein) column inside a microfluidic chip.[85] This allowed them to

detect the early stage cancer-diagnostic �FP in an automated system with an analysis time

of less than one hour. Xiao et al. have published a proof-of-principle using a microfluidic

chip with immobilized strands of DNA on it, able to selectively trap insulin and insuline-

like growth factor 2, with subsequently MALDI-TOF detection.[86] This work shows

great promise for the development of a more universal microfluidic chip-based approach

for the discovery of DNA-binding ligands. The recent move towards the use of magnetic

beads in affinity measurements combined with capillary electrophoresis can also lead to

interesting results. Adachi et al. for instance, immobilized bacterial 16S rRNA on

magnetic beads, and injected them into a capillary exposed to a magnetic field, thus

trapping the beads in the capillary.[87] Then using fluorescent probes, they are able to

quantify the varying strands of rRNA, as is shown in figure 1.8. Finally, Liu et al

developed teicoplanin-derivatized microbeads, that can be immobilized inside a

microfluidic chip with microscopic magnets embedded in the PDMS chip.[88] This

allows for the calculation of the binding constant for a natively fluorescent ligand to the

antibiotic teicoplanin.

When assessing the merits of affinity capillary electrophoresis, it is apparent the

field has evolved to a point where it can compete with all other affinity methods

described in this paper. It is a relatively simple and straightforward method to implement

and use routinely. However, it is unlikely ACE will evolve into a universal technique.

Due to limitations such as non-specific adsorption of the analytes to the capillary wall,

and Joule heating of the system, many targets are not suitable for analysis by ACE,

forcing the researcher to resort to other methodologies. Furthermore, implementation of

mass spectrometric detection is not as straightforward as in liquid chromatography, and

as a result, a great variety of detection methods is used that are less specific.

Chapter one

35

Figure 1.8: The concept of immobilized magnetic bead capillary electrophoresis: (a) RNA extraction,

biotynilation and immobilization on magnetic beads. (b) injection. (c) addition of

fluorescent label. (d) charging the capillary removing all compounds not trapped

magnetically (e) injection of formamide to release the label from the target RNA. (f)

detection of the dissociated label using laser induced fluorescence.

Chapter one

36

SPR-MS

Surface Plasmon Resonance (SPR) differs fundamentally from all other methods

presented in this review, because protein binding and detection takes place at the same

location. An SPR biosensor consists of a prism positioned against a thin metal layer

(often gold) and a solution at the opposite side of the metal layer. Surface plasmons will

occur at the interface between metal and solution, and these surface plasmons have a

certain wave vector, depending on the composition and roughness of the metal surface

and the composition of the solution. When a beam of light undergoes total internal

reflection at the prism-metal interface, the photons excite the electrons in the metal film.

At one specific angle of incidence, the wave vector of these excited electrons is equal to

that of the surface plasmons, resulting in a total energy transfer. This is the definition of

surface plasmon resonance and when it happens, the light beam is no longer reflected.

The biosensor mechanism depends on the fact that the angle at which this happens is

strongly dependent on the composition of the solution. When a ligand binds to a protein

immobilized on the metal sensor, this changes the wave vector of the surface plasmons,

and the angle of incidence at which surface plasmon resonance occurs. However, no

identification occurs, as a result of which SPR is nowadays oftentimes coupled to a mass

spectrometer in order to identify the binders.

Since the launch of commercially available SPR biosensors, such as the Biacore,

in 1993[89], surface plasmon resonance has become an increasingly prominent

technology in drug discovery. In the Biacore and most competing SPR biosensors, an

analyte is infused onto a chip with immobilized target protein, starts to bind until at a

certain moment all possible binding sites are occupied, and the chip is saturated. The

speed at which this happens is dependant upon association and dissociation rates of the

complex, completely analogous to frontal affinity chromatography. The resulting data

and data processing is also analogous to FAC. However, in SPR, the change in signal is

not dependant upon analyte concentration, but analyte molecular weight. This is why

SPR bioassays are especially suitable for the study of protein-protein interactions, or

other large molecules that bind to the immobilized target protein.

Chapter one

37

The first major applications of SPR biosensors were in the field of antibody-

antigen interactions[90], the study of DNA hybridization and dehybridization,[91] and

many other macromolecular interactions.[92] Around the same time, the first coupling of

SPR to MALDI-TOF mass spectrometry was reported, in which the SPR chip was

directly used as MALDI chip.[93,94]. Since that moment, many SPR-MALDI

methodologies have been developed, covering a wide assortment of target proteins.[95-

98]. A number of interesting reviews have been published providing an overview of the

importance of SPR-MS for modern-day proteomics.[99,100]

Interesting papers in recent SPR-MS method development include the work of

Borch et al. who exposed an immobilized enzyme to a possible inhibitor, and proceeded

to perform an activity assay by infusing the substrate and analyzing the substrate

conversion by MS.[101] Bouffartigues et al. published a high-throughput SPR-SELDI-

MS methodology able to identify protein bound to DNA.[102] Marchesini et al.

published an SPR methodology coupled online to a nano-HILIC-LC system, resulting in

an online electrospray interface between the SPR-LC and the mass spectrometer.[103]

In the field of small molecule research, significantly fewer methodologies have

been published, presumably due to the dependency of the SPR signal on molecular

weight and as a result, its improved performance for analytes above 15 kDa. An elegant

solution for analyzing small molecules is to immobilize the small molecules on the chip,

and subsequently measuring the binding of the target protein,[104] analogous to

immobilized antigens.[90] However, recently Jecklin et al. developed an SPR

methodology for seven analytes under 500 Da. Utilizing human carbonanhydrase

immobilized on the SPR chip, they managed to calculate accurate dissociation constants

for the testset, and compare them to two reference methods.[103] Also, Marchesini et al

successfully measured the binding and dissociation kinetics of a testset of seven low

molecular weight paralytic shellfish poisons binding to immobilized saxitoxin

monoclonal antibody (Figure 1.9).[105] Furthermore, recent publications by Wang et al.

and Miura et al. show the development of indirect SPR affinity methods, in which the

affinity of a small molecule is determined by its competition with a large ligand already

bound to the immobilized target protein.[106,107]

Chapter one

38

Figure 9: Binding and dissociation kinetics of seven paralytic shellfish poisons to

immobilized MAb GT13A. Reproduced from ref [105].

Concluding, SPR is an extremely powerful tool for protein affinity analysis, mainly

because of the quantification of the affinity of the analyte during it’s immobilization on

the SPR chip-surface. Its main drawback is that it is most efficient for compounds with a

high molecular weight, and thus most suitable for the study of protein-protein

interactions. However, developments that target small molecules are upcoming.

Conclusion

Reviewing the wide spectrum of methodologies discussed in this paper, all of

them can be separated into two general categories, with distinctly different applicability,

advantages and disadvantages. The first group consists of methods that immobilize the

target protein on a solid support, the second of methodologies that allow in solution

complexation. Ultrafiltration and size exclusion are solution based methods. The complex

is formed in solution, approaching in vivo circumstances as closely as possible. The same

goes for dynamic protein affinity selection and a number of capillary electrophoresis

Chapter one

39

methods. All other techniques: frontal affinity, zonal elution, affinity capture, most CE-

based methods and surface plasmon resonance use immobilized proteins.

When assessing the progress made using immobilized proteins, a number of

conclusions can be drawn. Frontal affinity chromatography will remain relevant, because

it is a cheap and easy to implement method to assess protein-ligand binding. The

immobilized target proteins are getting more and more complex, and none of the in-

solution based methods rivals the data resulting from FAC methodologies. The only

methodology that does is SPR. SPR-MS can produce the same affinity data as FAC, and

probably more. However, SPR is limited by its inability to measure small ligands

directly, and the more limited amount of target proteins that have proven to be

successfully immobilized on the SPR chip.

The solution-based methodologies might not provide the same amount of affinity

data, but they do not suffer from impaired binding properties due to protein

immobilization, and each of them has advantages of its own over the competition.

Ultrafiltration benefits from the reusability of the receptor, and the speed of

measurement. Capillary electrophoresis affinity selection can target proteins unsuitable

for any of the other methodologies, and will stay important despite issues with non-

specific absorption of the proteins to the capillary wall. Dynamic protein affinity

selection is very sensitive for weak binders, and suffers little from non-specific binding.

However, the size exclusion methodologies are the ones most commonly applied in

modern drug discovery for two simple reasons. SEC is very easy to implement and

automate using standard 96 well plates and equipment, and SEC is significantly faster

than all other techniques discussed.

As a result, when reviewing literature or visiting relevant conferences, most of the

research on affinity selection mass spectrometry in pharmaceutical industry is focused on

surface plasmon resonance technologies and size exclusion technologies. However, many

if not all of the discussed fields of affinity selection mass spectrometry will undoubtedly

result in new lead candidates in the upcoming years.

Chapter one

40

Reference List

1. Sinz A: Investigation of protein-ligand interactions by mass spectrometry. Chemmedchem 2007,

2:425-431.

2. Gingras AC, Gstaiger M, Raught B, Aebersold R: Analysis of protein complexes using mass

spectrometry. Nature Reviews Molecular Cell Biology 2007, 8:645-654.

3. Ferguson PL, Kuprowski MC, Boys BL, Wilson DJ, Pan JX, Konermann L: Protein Folding and

Protein-Ligand Interactions Monitored by Electrospray Mass Spectrometry. Current

Analytical Chemistry 2009, 5:186-204.

4. Nesatyy VJ: Mass spectrometry evaluation of the solution and gas-phase binding properties of

noncovalent protein complexes. International Journal of Mass Spectrometry 2002, 221:147-161.

5. de Jong LAA, Uges DRA, Franke JP, Bischoff R: Receptor-ligand binding assays: Technologies and

applications. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life

Sciences 2005, 829:1-25.

6. Cuatreca.P, Wilchek M, Anfinsen CB: SELECTIVE ENZYME PURIFICATION BY AFFINITY

CHROMATOGRAPHY. Proceedings of the National Academy of Sciences of the United States

of America 1968, 61:636-&.

7. Wofsy L, Burr B: USE OF AFFINITY CHROMATOGRAPHY FOR SPECIFIC PURIFICATION

OF ANTIBODIES AND ANTIGENS. Journal of Immunology 1969, 103:380-&.

8. Hage DS: Handbook of Affinity Chromatography 2006, 2nd Edition, Taylor and Francis, New York.

9. Hage DS: High-performance affinity chromatography: a powerful tool for studying serum protein

binding. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 2002, 768:3-30.

10. Ng ESM, Chan NWC, Lewis DF, Hindsgaul O, Schriemer DC: Frontal affinity chromatography-

mass spectrometry. Nature Protocols 2007, 2:1907-1917.

11. Pidgeon C, Venkataram UV: IMMOBILIZED ARTIFICIAL MEMBRANE

CHROMATOGRAPHY - SUPPORTS COMPOSED OF MEMBRANE-LIPIDS. Analytical

Biochemistry 1989, 176:36-47.

12. Beigi F, Wainer IW: Syntheses of immobilized G protein-coupled receptor chromatographic

stationary phases: Characterization of immobilized mu and kappa opioid receptors.

Analytical Chemistry 2003, 75:4480-4485.

13. Beigi F, Chakir K, Xiao RP, Wainer IW: G-protein-coupled receptor chromatographic stationary

phases. 2. Ligand-induced conformational mobility in an immobilized beta(2)-adrenergic receptor. Analytical Chemistry 2004, 76:7187-7193.

14. Temporini C, Ceruti S, Calleri E, Ferrario S, Moaddel R, Abbracchio MP, Massolini G: Development

of an immobilized GPR17 receptor stationary phase for binding determination using frontal

affinity chromatography coupled to mass spectrometry. Analytical Biochemistry 2009,

384:123-129.

15. Moaddel R, Wainer IW: The preparation and development of cellular membrane affinity

chromatography columns. Nature Protocols 2009, 4:197-205.

16. Besanger TR, Hodgson RJ, Guillon D, Brennan JD: Monolithic membrane-receptor columns:

Optimization of column performance for frontal affinity chromatography/mass

spectrometry applications. Analytica Chimica Acta 2006, 561:107-118.

17. Okanda FM, El Rassi Z: Affinity monolithic capillary columns for glycomics/proteomics: I.

Polymethacrylate monoliths with immobilized lectins for glycoprotein separation by affinity

capillary electrochromatography and affinity nano-liquid chromatography in either a single

column or columns coupled in series. Electrophoresis 2006, 27:1020-1030.

18. Feng S, Pan CS, Jiang XG, Xu SY, Zhou HJ, Ye ML, Zou HF: Fe3+ immobilized metal affinity

chromatography with silica monolithic capillary column for phosphoproteome analysis.

Proteomics 2007, 7:351-360.

19. Zhaoa XF, Zheng XH, Wei YM, Bian LJ, Wang SX, Zheng JB, Zhang YY, Li ZJ, Zang WJ:

Thermodynamic study of the interaction between terbutaline and salbutamol with an

immobilized beta(2)-adrenoceptor by high-performance liquid chromatography. Journal of

Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 2009, 877:911-

918.

Chapter one

41

20. Yoo MJ, Smith QR, Hage DS: Studies of imipramine binding to human serum albumin by high-

performance affinity chromatography. Journal of Chromatography B-Analytical Technologies

in the Biomedical and Life Sciences 2009, 877:1149-1154.

21. Bertucci C, Pistolozzi M, Felix G, Danielson UH: HSA binding of HIV protease inhibitors: a high-

performance affinity chromatography study. Journal of Separation Science 2009, 32:1625-

1631.

22. Huyer G, Kelly J, Moffat J, Zamboni R, Jia ZC, Gresser MJ, Ramachandran C: Affinity selection from

peptide libraries to determine substrate specificity of protein tyrosine phosphatases.

Analytical Biochemistry 1998, 258:19-30.

23. Manalan AS, Klee CB: AFFINITY SELECTION OF CHEMICALLY MODIFIED PROTEINS -

ROLE OF LYSYL RESIDUES IN THE BINDING OF CALMODULIN TO

CALCINEURIN. Biochemistry 1987, 26:1382-1390.

24. Kataoka K: HIGH-CAPACITY CELL-SEPARATION BY AFFINITY SELECTION ON

SYNTHETIC SOLID-PHASE MATRICES. Acs Symposium Series 1991, 464:159-174.

25. Bundy J, Fenselau C: Lectin-based affinity capture for MALDI-MS analysis of bacteria. Analytical

Chemistry 1999, 71:1460-1463.

26. Kaji H, Saito H, Yamauchi Y, Shinkawa T, Taoka M, Hirabayashi J, Kasai K, Takahashi N, Isobe T:

Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked

glycoproteins. Nature Biotechnology 2003, 21:667-672.

27. Krugmann S, Anderson KE, Ridley SH, Risso N, McGregor A, Coadwell J, Davidson K, Eguinoa A,

Ellson CD, Lipp P, et al.: Identification of ARAP3, a novel PI3K effector regulating both Arf

and Rho GTPases, by selective capture on phosphoinositide affinity matrices. Molecular Cell

2002, 9:95-108.

28. Kong XL, Huang LCL, Hsu CM, Chen WH, Han CC, Chang HC: High-affinity capture of proteins

by diamond nanoparticles for mass spectrometric analysis. Analytical Chemistry 2005,

77:259-265.

29. Ferrance JP: Gellan beads as a transparent media for protein immobilization and affinity capture.

Journal of Chromatography A 2007, 1165:86-92.

30. Freije JR, Bischoff R: Activity-based enrichment of matrix metalloproteinases using reversible

inhibitors as affinity ligands. Journal of Chromatography A 2003, 1009:155-169.

31. Freije R, Klein T, Ooms B, Kauffman HF, Bischoff R: An integrated high-performance liquid

chromatography-mass spectrometry system for the activity-dependent analysis of matrix metalloproteases. Journal of Chromatography A 2008, 1189:417-425.

32. Johnson EM, Ellis WR, Powers LS, Wysocki VH: Affinity Capture Mass Spectrometry of

Biomarker Proteins Using Peptide Ligands from Biopanning. Analytical Chemistry 2009,

81:5999-6005.

33. Gutierrez R, del Valle EMM, Galan MA: Immobilized metal-ion affinity chromatography: Status

and trends. Separation and Purification Reviews 2007, 36:71-111.

34. Cheeks MC, Kamal N, Sorrell A, Darling D, Farzaneh F, Slater NKH: Immobilized metal affinity

chromatography of histidine-tagged lentiviral vectors using monolithic adsorbents.

2009:2705-2711.

35. Jonker N, Kool J, Krabbe JG, Retra K, Lingeman H, Irth H: Screening of protein-ligand interactions

using dynamic protein-affinity chromatography solid-phase extraction-liquid chromatography-mass spectrometry. J Chromatogr A 2008, 1205:71-77.

36. Ahmed ARH, Olivier GWJ, Adams G, Erskine ME, Kinsman RG, Branch SK, Moss SH, Notarianni LJ,

Pouton CW: ISOLATION AND PARTIAL-PURIFICATION OF A MELANOCYTE-

STIMULATING HORMONE RECEPTOR FROM B16 MURINE MELANOMA-CELLS -

A NOVEL-APPROACH USING A CLEAVABLE BIOTINYLATED PHOTOACTIVATED

LIGAND AND STREPTAVIDIN-COATED MAGNETIC BEADS. Biochemical Journal

1992, 286:377-382.

37. Karlsson GB, Platt FM: ANALYSIS AND ISOLATION OF HUMAN TRANSFERRIN

RECEPTOR USING THE OKT-9 MONOCLONAL-ANTIBODY COVALENTLY CROSS-

LINKED TO MAGNETIC BEADS. Analytical Biochemistry 1991, 199:219-222.

38. Ji ZS, Pinon DI, Miller LJ: Development of magnetic beads for rapid and efficient metal-chelate

affinity purifications. Analytical Biochemistry 1996, 240:197-201.

Chapter one

42

39. Mehta RV, Upadhyay RV, Charles SW, Ramchand CN: Direct binding of protein to magnetic

particles. Biotechnology Techniques 1997, 11:493-496.

40. Jonker N, Kretschmer A, Kool J, Fernandez A, Kloos D, Krabbe JG, Lingeman H, Irth H: Online

Magnetic Bead Dynamic Protein-Affinity Selection Coupled to LC-MS for the Screening of

Pharmacologically Active Compounds. Analytical Chemistry 2009, 81:4263-4270.

41. Meyer A, Hansen DB, Gomes CSG, Hobley TJ, Thomas ORT, Franzreb M: Demonstration of a

strategy for product purification by high-gradient magnetic fishing: Recovery of superoxide

dismutase from unconditioned whey. Biotechnology Progress 2005, 21:244-254.

42. Marsza, x, x, P. M, Moaddel R, Kole S, Gandhari M, Bernier M, Wainer IW: Ligand and Protein

Fishing with Heat Shock Protein 90 Coated Magnetic Beads. Anal. Chem. 2008, 80:7571-7575.

43. Hu F, Deng C, Zhang X: Development of high performance liquid chromatography with

immobilized enzyme onto magnetic nanospheres for screening enzyme inhibitor. Journal of

Chromatography B 2008.

44. Whitlam JB, Brown KF: ULTRAFILTRATION IN SERUM-PROTEIN BINDING

DETERMINATIONS. Journal of Pharmaceutical Sciences 1981, 70:146-150.

45. Zhirkov YA, Piotrovskii VK: ON THE USEFULNESS OF ULTRAFILTRATION IN DRUG-

PROTEIN BINDING-STUDIES. Journal of Pharmacy and Pharmacology 1984, 36:844-845.

46. Wieboldt R, Zweigenbaum J, Henion J: Immunoaffinity ultrafiltration with ion spray HPLC/MS

for screening small-molecule libraries. Analytical Chemistry 1997, 69:1683-1691.

47. Weisiger RA, Ostrow JD, Koehler RK, Webster CC, Mukerjee P, Pascolo L, Tiribelli C: Affinity of

human serum albumin for bilirubin varies with albumin concentration and buffer composition - Results of a novel ultrafiltration method. Journal of Biological Chemistry 2001,

276:29953-29960.

48. Zlotos G, Oehlmann M, Nickel P, Holzgrabe U: Determination of protein binding of gyrase

inhibitors by means of continuous ultrafiltration. Pergamon-Elsevier Science Ltd: 1998:847-

858.

49. Kinawi A, Teller C: DETERMINATION OF DRUG-ALBUMIN BINDING IN BUFFERED

BOVINE SERUM-ALBUMIN SOLUTIONS APPLYING A MODIFIED

ULTRAFILTRATION PROCESS. Arzneimittel-Forschung/Drug Research 1979, 29-2:1495-

1500.

50. Heinze A, Holzgrabe U: Determination of the extent of protein binding of antibiotics by means of

an automated continuous ultrafiltration method. International Journal of Pharmaceutics 2006,

311:108-112.

51. Fung EN, Chen YH, Lau YY: Semi-automatic high-throughput determination of plasma protein

binding using a 96-well plate filtrate assembly and fast liquid chromatography-tandem mass

spectrometry. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life

Sciences 2003, 795:187-194.

52. Comess KM, Schurdak ME, Voorbach MJ, Coen M, Trumbull JD, Yang H, Gao L, Tang H, Cheng X,

Lerner CG, et al.: An ultraefficient affinity-based high-throughout screening process:

Application to bacterial cell wall biosynthesis enzyme MurF. Journal of Biomolecular

Screening 2006, 11:743-754.

53. Li HL, Song FR, Xing JP, Tsao R, Liu ZQ, Liu SY: Screening and Structural Characterization of

alpha-Glucosidase Inhibitors from Hawthorn Leaf Flavonoids Extract by Ultrafiltration LC-DAD-MSn and SORI-CID FTICR MS. Journal of the American Society for Mass Spectrometry

2009, 20:1496-1503.

54. vanBreemen RB, Huang CR, Nikolic D, Woodbury CP, Zhao YZ, Venton DL: Pulsed ultrafiltration

mass spectrometry: A new method for screening combinatorial libraries. Analytical

Chemistry 1997, 69:2159-2164.

55. Shin YG, Bolton JL, van Breemen RB: Screening drugs for metabolic stability using pulsed

ultrafiltration mass spectrometry. Combinatorial Chemistry & High Throughput Screening

2002, 5:59-64.

56. Cheng X, van Breemen RB: Mass spectrometry-based screening for inhibitors of beta-amyloid

protein aggregation. Analytical Chemistry 2005, 77:7012-7015.

57. Liu DT, Guo J, Luo Y, Broderick DJ, Schimerlik MI, Pezzuto JM, van Breemen RB: Screening for

ligands of human retinoid X receptor-alpha. Using ultrafiltration mass spectrometry.

Analytical Chemistry 2007, 79:9398-9402.

Chapter one

43

58. Moore JC: GEL PERMEATION CHROMATOGRAPHY .I. NEW METHOD FOR

MOLECULAR WEIGHT DISTRIBUTION OF HIGH POLYMERS. Journal of Polymer

Science Part a-General Papers 1964, 2:835-&.

59. Nath S, Brahma A, Bhattacharyya D: Extended application of gel-permeation chromatography by

spin column. Analytical Biochemistry 2003, 320:199-206.

60. Siegel MM, Tabei K, Bebernitz GA, Baum EZ: Rapid methods for screening low molecule mass

compounds non-covalently bound to proteins using size exclusion and mass spectrometry

applied to inhibitors of human cytomegalovirus protease. Journal of Mass Spectrometry 1998,

33:264-273.

61. Muckenschnabel I, Falchetto R, Mayr LM, Filipuzzi I: SpeedScreen: label-free liquid

chromatography-mass spectrometry-based high-throughput screening for the discovery of

orphan protein ligands. Analytical Biochemistry 2004, 324:241-249.

62. Brown N, Zehender H, Azzaoui K, Schuffenhauer A, Mayr LM, Jacoby E: A chemoinformatics

analysis of hit lists obtained from high-throughput affinity-selection screening. Journal of

Biomolecular Screening 2006, 11:123-130.

63. Annis DA, Athanasopoulos J, Curran PJ, Felsch JS, Kalghatgi K, Lee WH, Nash HM, Orminati JPA,

Rosner KE, Shipps GW, et al.: An affinity selection-mass spectrometry method for the

identification of small molecule ligands from self-encoded combinatorial libraries -

Discovery of a novel antagonist of E-coli dihydrofolate reductase. International Journal of

Mass Spectrometry 2004, 238:77-83.

64. Whitehurst CE, Nazef N, Annis DA, Hou YM, Murphy DM, Spacciapoli P, Yao ZP, Ziebell MR,

Cheng CC, Shipps GW, et al.: Discovery and characterization of orthosteric and allosteric

muscarinic M-2 acetylcholine receptor ligands by affinity selection-mass spectrometry.

Journal of Biomolecular Screening 2006, 11:194-207.

65. Annis DA, Cheng CC, Chuang C-C, McCarter JD, Nash HM, Nazef N, Rowe T, Kurzeja RJM, Shipps

GW, Jr.: Inhibitors of the lipid phosphatase SHIP2 discovered by high-throughput affinity

selection-mass spectrometry screening of combinatorial libraries. Comb Chem High

Throughput Screen 2009, 12:760-771.

66. Cheng CC, Shipps GW, Yang Z, Sun B, Kawahata N, Soucy KA, Soriano A, Orth P, Xiao L, Mann P,

et al.: Discovery and optimization of antibacterial AccC inhibitors. Bioorg Med Chem Lett

2009, 19:6507-6514.

67. Whitehurst CE, Annis DA: Affinity selection-mass spectrometry and its emerging application to

the high throughput screening of G protein-coupled receptors. Combinatorial Chemistry &

High Throughput Screening 2008, 11:427-438.

68. Flarakos J, Morand KL, Vouros P: High-throughput solution-based medicinal library screening

against human serum albumin. Analytical Chemistry 2005, 77:1345-1353.

69. Schmidt AC, Fahlbusch B, Otto M: Size exclusion chromatography coupled to electrospray

ionization mass spectrometry for analysis and quantitative characterization of arsenic

interactions with peptides and proteins. Journal of Mass Spectrometry 2009, 44:898-910.

70. Wolf C, Strenziok R, Kyriakopoulos A: Elevated metallothionein-bound cadmium concentrations

in urine from bladder carcinoma patients, investigated by size exclusion chromatography-

inductively coupled plasma mass spectrometry. Analytica Chimica Acta 2009, 631:218-222.

71. Adam GC, Parish CA, Wisniewski D, Meng JC, Liu M, Calati K, Stein BD, Athanasopoulos J,

Liberator P, Roemer T, et al.: Application of Affinity Selection/Mass Spectrometry to

Determine the Structural Isomer of Parnafungins Responsible for Binding Polyadenosine

Polymerase. Journal of the American Chemical Society 2008, 130:16704-16710.

72. Kraak JC, Busch S, Poppe H: STUDY OF PROTEIN DRUG-BINDING USING CAPILLARY

ZONE ELECTROPHORESIS. Elsevier Science Bv: 1992:257-264.

73. Baba Y, Tsuhako M, Sawa T, Akashi M, Yashima E: SPECIFIC BASE RECOGNITION OF

OLIGODEOXYNUCLEOTIDES BY CAPILLARY AFFINITY GEL-

ELECTROPHORESIS USING POLYACRYLAMIDE POLY(9-VINYLADENINE)

CONJUGATED GEL. Analytical Chemistry 1992, 64:1920-1925.

74. Chen Z, Weber SG: Determination of binding constants by affinity capillary electrophoresis,

electrospray ionization mass spectrometry and phase-distribution methods. Trac-Trends in

Analytical Chemistry 2008, 27:738-748.

Chapter one

44

75. Liu XJ, Dahdouh F, Salgado M, Gomez FA: Recent Advances in Affinity Capillary Electrophoresis

(2007). Journal of Pharmaceutical Sciences 2009, 98:394-410.

76. Fermas S, Gonnet F, Varenne A, Gareil P, Daniel R: Frontal analysis capillary electrophoresis

hyphenated to electrospray ionization mass spectrometry for the characterization of the

antithrombin/heparin pentasaccharide complex. Analytical Chemistry 2007, 79:4987-4993.

77. Mrestani Y, Hammitzch M, Neubert RHH: Investigation of the Interaction Between Lidocaine and

the Components of Hyaluronic Acid Using Frontal Analysis Continuous Capillary

Electrophoresis. Chromatographia 2009, 69:1321-1324.

78. Franzen U, Jorgensen L, Larsen C, Heegaard NHH, Ostergaard J: Determination of liposome-buffer

distribution coefficients of charged drugs by capillary electrophoresis frontal analysis.

Electrophoresis 2009, 30:2711-2719.

79. Liu XM, Li SY, Zhang JS, Chen XG: Flow injection-capillary electrophoresis frontal analysis

method for the study of the interactions of a series of drugs with human serum albumin.

Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 2009,

877:3144-3150.

80. Amini A, Westerlund D: Evaluation of association constants between drug enantiomers and

human alpha(1)-acid glycoprotein by applying a partial-filling technique in affinity capillary

electrophoresis. Analytical Chemistry 1998, 70:1425-1430.

81. Villareal V, Kaddis J, Azad M, Zurita C, Silva I, Hernandez L, Rudolph M, Moran J, Gomez FA:

Partial-filling affinity capillary electrophoresis. Analytical and Bioanalytical Chemistry 2003,

376:822-831.

82. Tanaka Y, Terabe S: Separation of the enantiomers of basic drugs by affinity capillary

electrophoresis using a partial filling technique and alpha(1)-acid glycoprotein as chiral

selector. Chromatographia 1997, 44:119-128.

83. Zavaleta J, Chinchilla DB, Ramirez A, Pao A, Martinez K, Nilapwar S, Ladbury JE, Mallik S, Gomez

FA: Partial filling multiple injection affinity capillary electrophoresis (PFMIACE) to

estimate binding constants of receptors to ligands. Talanta 2007, 71:192-201.

84. Almeda S, Salinas E, Arce C, Moreno A, Arce L, Valcarcel M: Use of multiple sequential injections

of equal volumes to determine the apparent binding constant for antibody-antigen

complexes by capillary electrophoresis. Talanta 2009, 78:1446-1451.

85. Yang WC, Sun XH, Wang HY, Woolley AT: Integrated Microfluidic Device for Serum Biomarker

Quantitation Using Either Standard Addition or a Calibration Curve. Analytical Chemistry 2009, 81:8230-8235.

86. Xiao JF, Carter JA, Frederick KA, McGown LB: A genome-inspired DNA ligand for the affinity

capture of insulin and insulin-like growth factor-2. Journal of Separation Science 2009,

32:1654-1664.

87. Adachi K, Yamaguchi M, Nakashige M, Kanagawa T, Torimura M, Tsuneda S, Sekiguchi Y, Noda N:

Affinity capillary electrophoresis with magnetic beads for multiplex quantitative analysis of

bacterial 16S rRNA. Journal of Bioscience and Bioengineering 2009, 107:662-667.

88. Liu XJ, Gomez FA: Microchip frontal affinity chromatography to study the binding of a ligand to

teicoplanin-derivatized microbeads. Electrophoresis 2009, 30:1194-1197.

89. Oshannessy DJ, Brighamburke M, Peck K: IMMOBILIZATION CHEMISTRIES SUITABLE FOR

USE IN THE BIACORE SURFACE-PLASMON RESONANCE DETECTOR. Analytical Biochemistry 1992, 205:132-136.

90. Malmqvist M: SURFACE-PLASMON RESONANCE FOR DETECTION AND

MEASUREMENT OF ANTIBODY ANTIGEN AFFINITY AND KINETICS. Current

Opinion in Immunology 1993, 5:282-286.

91. Peterlinz KA, Georgiadis RM, Herne TM, Tarlov MJ: Observation of hybridization and

dehybridization of thiol-tethered DNA using two-color surface plasmon resonance

spectroscopy. Journal of the American Chemical Society 1997, 119:3401-3402.

92. Karlsson R, Falt A: Experimental design for kinetic analysis of protein-protein interactions with

surface plasmon resonance biosensors. Journal of Immunological Methods 1997, 200:121-133.

93. Nelson RW, Krone JR, Jansson O: Surface plasmon resonance biomolecular interaction analysis

mass spectrometry .1. Chip-based analysis. Analytical Chemistry 1997, 69:4363-4368.

94. Nelson RW, Krone JR, Jansson O: Surface plasmon resonance biomolecular interaction analysis

mass spectrometry .2. Fiber optic based analysis. Analytical Chemistry 1997, 69:4369-4374.

Chapter one

45

95. Zhukov A, Schurenberg M, Jansson O, Areskoug D, Buijs J: Integration of surface plasmon

resonance with mass spectrometry: automated ligand fishing and sample preparation for

MALDI MS using a Biacore 3000 biosensor. J Biomol Tech 2004, 15:112-119.

96. Grote J, Dankbar N, Gedig E, Koenig S: Surface plasmon resonance/mass spectrometry interface.

Analytical Chemistry 2005, 77:1157-1162.

97. Nedelkov D: Development of surface plasmon resonance mass spectrometry array platform.

Analytical Chemistry 2007, 79:5987-5990.

98. Bellon S, Buchmann W, Gonnet F, Jarroux N, Anger-Leroy M, Guillonneau F, Daniel R: Hyphenation

of Surface Plasmon Resonance Imaging to Matrix-Assisted Laser Desorption Ionization

Mass Spectrometry by On-Chip Mass Spectrometry and Tandem Mass Spectrometry

Analysis. Analytical Chemistry 2009, 81:7695-7702.

99. Nedelkov D, Nelson RW: Surface plasmon resonance mass spectrometry: recent progress and

outlooks. Trends in Biotechnology 2003, 21:301-305.

100. Visser NFC, Heck AJR: Surface plasmon resonance mass spectrometry in proteomics. Expert

Review of Proteomics 2008, 5:425-433.

101. Borch J, Roepstorff P: Screening for enzyme inhibitors by surface plasmon resonance combined

with mass spectrometry. Analytical Chemistry 2004, 76:5243-5248.

102. Bouffartigues E, Leh H, Anger-Leroy M, Rimsky S, Buckle M: Rapid coupling of Surface Plasmon

Resonance (SPR and SPRi) and ProteinChip (TM) based mass spectrometry for the

identification of proteins in nucleoprotein interactions. Nucleic Acids Research 2007, 35.

103. Jecklin MC, Schauer S, Dumelin CE, Zenobi R: Label-free determination of protein-ligand binding

constants using mass spectrometry and validation using surface plasmon resonance and

isothermal titration calorimetry. Journal of Molecular Recognition 2009, 22:319-329.

104. Visser NFC, Scholten A, van den Heuvel RHH, Heck AJR: Surface-plasmon-resonance-based

chemical proteomics: Efficient specific extraction and semiquantitative identification of

cyclic nucleotide-binding proteins from cellular lysates by using a combination of surface

plasmon resonance, sequential elution and liquid chromatography-tandem mass

spectrometry. Chembiochem 2007, 8:298-305.

105. Marchesini GR, Hooijerink H, Haasnoot W, Buijs J, Campbell K, Elliott CT, Nielen MWF: Towards

Surface Plasmon Resonance biosensing combined with bioaffinity-assisted nano HILIC

Liquid Chromatography Time-of-flight Mass Spectrometry identification of Paralytic

Shellfish Poisons. Trac-Trends in Analytical Chemistry 2009, 28:792-803.

106. Wang JL, Munir A, Zhou HS: Au NPs-aptamer conjugates as a powerful competitive reagent for

ultrasensitive detection of small molecules by surface plasmon resonance spectroscopy.

Talanta 2009, 79:72-76.

107. Miura N, Shankaran DR, Gobi KV, Kawaguchi T, Kim SJ: An Overview of the Development and

Application of Surface Plasmon Resonance Based Immunosensors for Detection of Small

Molecules. Sensor Letters 2008, 6:891-902.

108. Inglese J, Johnson RL, Simeonov A, Xia MH, Zheng W, Austin CP, Auld DS: High-throughput

screening assays for the identification of chemical probes. Nature Chemical Biology 2007,

3:466-479.

109. Li ZY, Mehdi S, Patel I, Kawooya J, Judkins M, Zhang WH, Diener K, Lozada A, Dunnington D: An

ultra-high throughput screening approach for an adenine transferase using fluorescence polarization. Journal of Biomolecular Screening 2000, 5:31-37.

110. Parker GJ, Law TL, Lenoch FJ, Bolger RE: Development of high throughput screening assays

using fluorescence polarization: Nuclear receptor-ligand-binding and kinase/phosphatase

assays. Journal of Biomolecular Screening 2000, 5:77-88.

111. Yan B, Fang LL, Irving M, Zhang S, Boldi AM, Woolard F, Johnson CR, Kshirsagar T, Figliozzi GM,

Krueger CA, et al.: Quality control in combinatorial chemistry: Determination of the

quantity, purity, and quantitative purity of compounds in combinatorial libraries. Journal of

Combinatorial Chemistry 2003, 5:547-559.

112. Hofstadler SA, Sannes-Lowery KA: Applications of ESI-MS in drug discovery: interrogation of

noncovalent complexes. Nature Reviews Drug Discovery 2006, 5:585-595.

113. Cunningham BT, Li P, Schulz S, Lin B, Baird C, Gerstenmaier J, Genick C, Wang F, Fine E, Laing L:

Label-free assays on the BIND system. Journal of Biomolecular Screening 2004, 9:481-490.

Chapter one

46

114. Hamalainen MD, Zhukov A, Ivarsson M, Fex T, Gottfries J, Karlsson R, Bjorsne M: Label-free

primary screening and affinity ranking of fragment libraries using parallel analysis of

protein panels. Journal of Biomolecular Screening 2008, 13:202-209.

115. Moaddel R, Calleri E, Massolini G, Frazier CR, Wainer IW: The synthesis and initial

characterization of an immobilized purinergic receptor (P2Y(1)) liquid chromatography

stationary phase for online screening. Analytical Biochemistry 2007, 364:216-218.

116. Ng W, Dai JR, Slon-Usakiewicz JJ, Redden PR, Pasternak A, Reid N: Automated multiple ligand

screening by frontal affinity chromatography-mass spectrometry (FAC-MS). Journal of

Biomolecular Screening 2007, 12:167-174.

117. Schlosser G, Vekey K, Malorni A, Pocsfalvi G: Combination of solid-phase affinity capture on

magnetic beads and mass spectrometry to study non-covalent interactions: example of minor

groove binding drugs. Rapid Communications in Mass Spectrometry 2005, 19:3307-3314.

118. Comess KM, Trumbull JD, Park C, Chen ZH, Judge RA, Voorbach MJ, Coen M, Gao L, Tang H,

Kovar P, et al.: Kinase drug discovery by affinity selection/mass spectrometry (ASMS):

Application to DNA damage checkpoint kinase Chk1. Journal of Biomolecular Screening

2006, 11:755-764.

119. Van Breemen RB, Huang CR, Nikolic D, Woodbury CP, Zhao YZ, Venton DL: Pulsed

ultrafiltration mass spectrometry: A new method for screening combinatorial libraries.

Analytical Chemistry 1997, 69:2159-2164.

120. Katzenellenbogen BS, Choi IH, Delage-Mourroux R, Ediger TR, Martini PGV, Montano M, Sun J,

Weis K, Katzenellenbogen JA: Molecular mechanisms of estrogen action: selective ligands

and receptor pharmacology. Journal of Steroid Biochemistry and Molecular Biology 2000,

74:279-285.

121. Knox AJS, Meegan MJ, Lloyd DG: Estrogen receptors: Molecular interactions, virtual screening

and future prospects. Current Topics in Medicinal Chemistry 2006, 6:217-243.

122. Rich RL, Hoth LR, Geoghegan KF, Brown TA, LeMotte PK, Simons SP, Hensley P, Myszka DG:

Kinetic analysis of estrogen receptor/ligand interactions. Proceedings of the National Academy

of Sciences of the United States of America 2002, 99:8562-8567.

123. Ali S, Coombes RC: Estrogen receptor alpha in human breast cancer: occurrence and

significance. J Mammary Gland Biol Neoplasia 2000, 5:271-281.

124. Sano M, Inoue S, Hosoi T, Ouchi Y, Emi M, Shiraki M, Orimo H: ASSOCIATION OF

ESTROGEN-RECEPTOR DINUCLEOTIDE REPEAT POLYMORPHISM WITH OSTEOPOROSIS. Biochemical and Biophysical Research Communications 1995, 217:378-383.

125. Oosterkamp AJ, Hock B, Seifert M, Irth H: Novel monitoring strategies for xenoestrogens. Trac-

Trends in Analytical Chemistry 1997, 16:544-553.

126. Seifert M, Brenner-Weiss G, Haindl S, Nusser M, Obst U, Hock B: A new concept for the bioeffects-

related analysis of xenoestrogens: Hyphenation of receptor assays with LC-MS. Fresenius

Journal of Analytical Chemistry 1999, 363:767-770.

127. Kool J, Ramautar R, van Liempd SM, Beckman J, de Kanter FJJ, Meerman JHN, Schenk T, Irth H,

Commandeur JNM, Vermeulen NPE: Rapid on-line profiling of estrogen receptor binding

metabolites of tamoxifen. Journal of Medicinal Chemistry 2006, 49:3287-3292.

128. Schobel U, Frenay M, Van Elswijk DA, McAndrews JM, Long KR, Olson LM, Bobzin SC, Irth H:

High resolution screening of plant natural product extracts for estrogen receptor alpha and beta binding activity using an online HPLC-MS biochemical detection system. Journal of

Biomolecular Screening 2001, 6:291-303.

129. Choi Y, van Breemen RB: Development of a screening assay for Ligands to the estrogen receptor

based on magnetic microparticles and LC-MS. Combinatorial Chemistry & High Throughput

Screening 2008, 11:1-6.

130. Lewis JS, Jordan VC: Selective estrogen receptor modulators (SERMs): Mechanisms of

anticarcinogenesis and drug resistance. Mutation Research-Fundamental and Molecular

Mechanisms of Mutagenesis 2005, 591:247-263.

131. Blair RM, Fang H, Branham WS, Hass BS, Dial SL, Moland CL, Tong WD, Shi LM, Perkins R,

Sheehan DM: The estrogen receptor relative binding affinities of 188 natural and

xenochemicals: Structural diversity of ligands. Toxicological Sciences 2000, 54:138-153.

132. Eiler S, Gangloff M, Duclaud S, Moras D, Ruff M: Overexpression, purification, and crystal

structure of native ER alpha LBD. Protein Expression and Purification 2001, 22:165-173.

Chapter one

47

133. Hu F, Zhang H, Lin H, Deng C, Zhang X: Enzyme Inhibitor Screening by Electrospray Mass

Spectrometry with Immobilized Enzyme on Magnetic Silica Microspheres. Journal of the

American Society of Mass Spectrometry 2008.

134. Moaddel R, Marszall MP, Bighi F, Yang Q, Duan X, Wainer IW: Automated Ligand Fishing Using

Human Serum Albumin-Coated Magnetic Beads. Anal. Chem. 2007, 79:5414-5417.

135. Luckarift HR, Johnson GR, Spain JC: Silica-immobilized enzyme reactors; application to

cholinesterase-inhibition studies. Journal of Chromatography B 2006, 843:310-316.

136. Moaddel R, Lu LL, Baynham M, Wainer IW: Immobilized receptor- and transporter-based liquid

chromatographic phases for on-line pharmacological and biochemical studies: a mini-review. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life

Sciences 2002, 768:41-53.

137. Moaddel R, Price GB, Juteau JM, Leffak M, Wainer IW: The synthesis and initial characterization

of an immobilized DNA unwinding element binding (DUE-B) protein chromatographic

stationary phase. Journal of Chromatography B-Analytical Technologies in the Biomedical and

Life Sciences 2005, 820:197-203.

138. Yao N, Chen HM, Lin HQ, Deng CH, Zhang XM: Enrichment of peptides in serum by C-8-

functionalized magnetic nanoparticles for direct matrix-assisted laser desorption/ionization

time-of-flight mass spectrometry analysis. Journal of Chromatography A 2008, 1185:93-101.

139. Baumann S, Ceglarek U, Fiedler GM, Lembcke J, Leichtle A, Thiery J: Standardized approach to

proteome profiling of human serum based on magnetic bead separation and matrix-assisted

laser desorption/ionization time-of-flight mass spectrometry. Clinical Chemistry 2005, 51:973-

980.

140. Zhang RQ, Hirakawa K, Seto D, Soh N, Nakano K, Masadome T, Nagata K, Sakamoto K, Imato T:

Sequential injection chemiluminescence immunoassay for anionic surfactants using

magnetic microbeads immobilized with an antibody. Talanta 2005, 68:231-238.

141. Kourilov V, Steinitz M: Magnetic-bead enzyme-linked immunosorbent assay verifies adsorption

of ligand and epitope accessibility. Analytical Biochemistry 2002, 311:166-170.

142. Hayes MA, Polson NA, Phayre AN, Garcia AA: Flow-based microimmunoassay. Analytical

Chemistry 2001, 73:5896-5902.

143. Odabas S, Sayar F, Güven G, Yanikkaya-Demirel G, Piskin E: Separation of mesenchymal stem

cells with magnetic nanosorbents carrying CD105 and CD73 antibodies in flow-through and

batch systems. Journal of Chromatography B 2007:74-80.

144. Girault S, Chassaing G, Blais JC, Brunot A, Bolbach G: Coupling of MALDI-TOF mass analysis to

the separation of biotinylated peptides by magnetic streptavidin beads. Analytical Chemistry

1996, 68:2122-2126.

145. Wang JH, Hansen EH, Miro M: Sequential injection-bead injection-lab-on-valve schemes for on-

line solid phase extraction and preconcentration of ultra-trace levels of heavy metals with

determination by electrothermal atomic absorption spectrometry and inductively coupled

plasma mass spectrometry. Analytica Chimica Acta 2003, 499:139-147.

146. Verpoorte E: Beads and chips: new recipes for analysis. Lab on a Chip 2003, 3:60N-68N.

147. Kozeny J: Ueber kapillare Leitung des Wassers im Boden. Wiener Akademie der Wissenschaften

1927, 136:271.

148. Carman PC: The determination of the specific surface of powders. J. Soc. Chem. Ind. Trans. 1938,

57:225.

149. De Smet J, Gzil P, Vervoort N, Verelst H, Baron GV, Desmet G: On the optimisation of the bed

porosity and the particle shape of ordered chromatographic separation media. Journal of

Chromatography A 2005, 1073:43-51.

150. Elders N, van der Born D, Hendrickx LJD, Timmer BJJ, Krause A, Janssen E, de Kanter FJJ, Ruijter

E, Orru RVA: The Efficient One-Pot Reaction of up to Eight Components by the Union of

Multicomponent Reactions. Angewandte Chemie-International Edition 2009, 48:5856-5859.

151. Groenendaal B, Ruijter E, Orru RVA: 1-Azadienes in cycloaddition and multicomponent reactions

towards N-heterocycles. Chemical Communications 2008:5474-5489.

152. Scheffelaar R, Nijenhuis RAK, Paravidino M, Lutz M, Spek AL, Ehlers AW, de Kanter FJJ, Groen

MB, Orru RVA, Ruijter E: Synthesis of Conformationally Constrained Peptidomimetics using

Multicomponent Reactions. Journal of Organic Chemistry 2009, 74:660-668.

Chapter one

48

153. Gust R, Keilitz R, Schmidt K, von Rauch M: (4R,5S)/(4S,5R)-4,5-Bis(4-hydroxyphenyl)-2-

imidazolines: Ligands for the estrogen receptor with a novel binding mode. Journal of

Medicinal Chemistry 2002, 45:3356-3365.

154. Laios I, Cleeren A, Leclercq G, Nonclercq D, Laurent G, Schlenk M, Wellner A, Gust R: Effects of

(R,S)/(S,R),4,5-bis(2-chloro-4-hydroxyphenyl)-2-imidazolines and (R,S)/(S,R)-2,3-bis (2-

chloro-4-hydroxyphenyl)piperazines on estrogen receptor alpha level and transcriptional

activity in MCF-7 cells. Biochemical Pharmacology 2007, 74:1029-1038.

155. von Rauch M, Schlenk M, Gust R: Effects of C2-alkylation, N-alkylation, and N,N '-dialkylation

on the stability and estrogen receptor interaction of (4R,5S)/(4S,5R)-4,5-bis(4-hydroxyphenyl)-2-imidazolines. Journal of Medicinal Chemistry 2004, 47:915-927.

156. Groenendaal B, Ruijter E, de Kanter FJJ, Lutz M, Spek AL, Orru RVA: Generation of molecular

diversity using a complexity-generating MCR-platform towards triazinane diones. Organic

& Biomolecular Chemistry 2008, 6:3158-3165.

157. Elders N, Ruijter E, de Kanter FJJ, Janssen E, Lutz M, Spek AL, Orru RVA: A Multicomponent

Reaction Towards N-(Cyanomethyl)amides. Chemistry-a European Journal 2009, 15:6096-

6099.

158. Elders N, Ruijter E, de Kanter FJJ, Groen MB, Orru RVA: Selective formation of 2-imidazolines

and 2-substituted oxazoles by using a three-component reaction. Chemistry-a European

Journal 2008, 14:4961-4973.

159. Groenendaal B, Vugts DJ, Schmitz RF, de Kanter FJJ, Ruijter E, Groen MB, Orru RVA: A

multicomponent synthesis of triazinane diones. Journal of Organic Chemistry 2008, 73:719-

722.

160. Paravidino M, Bon RS, Scheffelaar R, Vugts DJ, Znabet A, Schmitz RF, de Kanter FJJ, Lutz M, Spek

AL, Groen MB, et al.: Diastereoselective multicomponent synthesis of dihydropyridones with

an isocyanide functionality. Organic Letters 2006, 8:5369-5372.

161. Glasnov TN, Vugts DJ, Koningstein MM, Desai B, Fabian WMF, Orru RVA, Kappe CO:

Microwave-assisted Dimroth rearrangement of thiazines to dihydropyrimidinethiones:

Synthetic and mechanistic aspects. Qsar & Combinatorial Science 2006, 25:509-518.

162. Scheffelaar R, Paravidino M, Muilwijk D, Lutz M, Spek AL, de Kanter FJJ, Orru RVA, Ruijter E: A

Novel Three-Component Reaction toward Dihydrooxazolopyridines. Organic Letters 2009,

11:125-128.