Identification of Posttranslational Modifications For Sample Prep.

Identification of Posttranslational ModificationsFor Sample Prep

Complexity of the Proteome

Protein processing and modification comprise an important third dimension of information, beyond those of DNA sequence and protein sequence.

The thousands of component proteins of a cell and their post-translational modifications may change with the cell cycle, environmental conditions, developmental stage, and metabolic state.

Proteomic approaches that don’t just identify proteins but also find their post-translational modifications are needed!

Post-translational Modification

• What purpose ?

- targeting (eg. some lipoproteins)

- stability (eg. secreted glycoproteins )

- function (eg. surface glycoproteins)

- control of activity (eg. clotting factors, caspases)

• How can we study it ?

Definitions of the components:

1. Post-translational modification (PTM): The chemical modifications that take place at certain amino acid residues after the protein is synthesized by translation are known as post-translational modifications. These are essential for normal functioning of the protein. Some of the most commonly observed PTMs include:

a) Phosphorylation: The process by which a phosphate group is attached to certain amino acid side chains in the

protein, most commonly serine, threonine and tyrosine.

b) Glycosylation: The attachment of sugar moieties to nitrogen or oxygen atoms present in the side chains of amino

acids like aspargine, serine or threonine.

c) Acylation: The process by which an acyl group is linked to the side chain of amino acids like asparagine, glutamine or

lysine.

d) Alkylation: Addition of alkyl groups, most commonly a methyl group to amino acids such as lysine or arginine. Other longer chain alkyl groups may also be attached in some cases.

e) Hydroxylation: This PTM is most often found on proline and lysine residues which make up the collagen tissue. It

enables crosslinking and therefore strengthening of the muscle fibres.

Definitions of the components

2. Protein translation: The process by which the mRNA template is read by ribosomes to synthesize the corresponding protein molecule on the basis of the three letter codons, which code for specific amino acids.

3. Cytosol: A cellular compartment that serves as the site for protein synthesis.

4. Signal sequence: A sequence that helps in directing the newly synthesized polypeptide chain to its appropriate intracellular organelle. This sequence is most often cleaved following protein folding and PTM.

5. Endoplasmic reticulum: A membrane-bound cellular organelle that acts as a site for post-translational modification of the newly synthesized polypeptide chains.

6. Cleaved protein: The protein product obtained after removal of certain amino acid sequences such as N- or C-terminal sequences, signal sequence etc.

Proteomic analysis of PTMs

Mann and Jensen, Nature Biotech. 21, 255 (2003)

8

Adduct formation – expect the unexpected

Adduct ion Percent [%] Adduct ion Percent [%] Adduct ion Percent [%] Adduct ion Percent [%] Adduct ion Percent [%][M+H]+ 62.55381 [M+H-C3H8O]+ 0.02667 [M-CCl3]+ 0.00381 [M(37Cl)]+. 0.00190 [M-2H+Na]- 0.00127

[M+2H]2+ 11.44459 [M-H-H2O-CO2]- 0.02667 [M-H-CO2]- 0.00381 [M-CH3]+ 0.00190 [M-H+Co]+ 0.00127

[M+H-H2O]+ 8.77598 [M-H-H2O-HCO2H]- 0.02667 [M+H-C5H7PO6]+ 0.00381 [M+H-C4H11N]+ 0.00190 [M+H-(CH3)2NH-C3H6]+ 0.00127

[M-H]- 6.25214 [M+H-3H2O]+ 0.02540 [M+H-HCl]+ 0.00381 [M+H-NO2-CHO]+ 0.00190 [M+H-C10H6(OH)N]+ 0.00127

[M+Na]+ 5.51055 [M+H-CHN]+ 0.02540 [M+H-C12H12N2O3]+ 0.00381 [M-H-HF]- 0.00190 [M-H+Ni]+ 0.00127

[M+H-NH3]+ 1.19494 [M+K-3H]2- 0.01905 [M+H-CH3CO2H]+ 0.00381 [M(37Cl)+H]+ 0.00190 [M-H-H2O-C4H7CO2H]- 0.00127

[M+NH4]+ 0.73715 [M+H-(CH3)2NH]+ 0.01524 [M+H-CH3]+. 0.00381 [M-H-C6H10O5]- 0.00190 [M+H-OH]+ 0.00127

[M-H-H2O]- 0.34604 [M+H-CHNO]+ 0.01333 [M+H-H2]+ 0.00381 [M+H-H2O-C6H13N]+ 0.00190 [M(81Br)+H]+... 0.00127

[M-H+2Na]+ 0.32953 [M+H-C2H6O]+ 0.01333 [M+H-C3H8NO6P]+ 0.00317 [M+H-H2O-H3PO4]+ 0.00190 [M-H-CH2O-CH2NH]- 0.00127

[M-H+H2O]- 0.24508 [M+H-CH4O]+ 0.01270 [M+H-C5H14NO4P]+ 0.00317 [M+H-C5H7PO6-NH3]+ 0.00190 [M+H-CO-CONH]+ 0.00127

[M+NH4-H2O]+ 0.22984 [M+H-C7H13NO3]+ 0.01143 [M+Li-(CH3)3N]+ 0.00317 [M-H-C5H7PO6]- 0.00190 [M-H-CONH]- 0.00127

[M+H+H2O]+ 0.19429 [M+Na-2H]- 0.00952 [M+Li-C5H14NO4P]+ 0.00317 [M+H-H2S]+ 0.00190 [M+H-C3H4O2]+ 0.00127

[M+H+Na]2+ 0.18286 [M-H-CH2O]- 0.00952 [M+Cl]- 0.00317 [M+H-H2O-C8H8]+ 0.00190 [M+H-C3H6O4]+ 0.00127

[M+H+K]2+ 0.17524 [M+H-C11H12N2O3]+ 0.00952 [M(35Cl)-H]- 0.00317 [M+H-H2O-NH3-C8H8]+ 0.00190 [M+Na-H2S]+ 0.00127

[M-2H]2- 0.13968 [M+H-C13H16N3O4]+ 0.00952 [M(37Cl)-H]- 0.00317 [M+H-H2O-NH3-C8H8-CO]+ 0.00190 [M-H+2Na-H2S]+ 0.00127

[M+2Na]2+ 0.13778 [M+H-C17H25N3O4]+ 0.00952 [M-H-C5H7O6P]- 0.00317 [M+H-H2O-NH3]+ 0.00190 [M-C5H5Cl]+ 0.00127

[M+2H-NH3]2+ 0.13714 [M+CH3CO2]- 0.00889 [M+H-C3H7O5P]+ 0.00317 [M+H-C3H6]+ 0.00190 [M+H-N2]+ 0.00127

[M+K]+ 0.13651 [M-H2O+Na]+ 0.00825 [M-H-C6H6N8O]- 0.00317 [M+HCO2-320]- 0.00190 [M+H-H2O-CO]+ 0.00127

[M+H-2H2O]+ 0.11810 [M-H+NH3]- 0.00762 [M(81Br)+H]+ 0.00317 [M+H-C3H7N]+ 0.00190 [M-H-H3PO4]- 0.00127

[M+3H]3+ 0.06667 [M+H-C9H9NO]+ 0.00762 [M-C4H9]+ 0.00317 [M-H-H2]- 0.00190 [M+H+CH3CN]+ 0.00127

[M+2H-H2O]2+ 0.06476 [M+H-C15H21N2O3]+ 0.00762 [M-2H+3Li]+ 0.00254 [M-H-C16H30O-H2O]- 0.00190 [M+H-C4H6]+ 0.00127

[M]+. 0.05905 [M-2H+3Na]+ 0.00698 [M-H-HCl]- 0.00254 [M-H-CH4O]- 0.00190 [M+H-CH3OH]+ 0.00127

[M+2Na-H]+ 0.05143 [M+HCO2]- 0.00635 [M+2Li-H]+ 0.00254 [M+H-C10H8FN3]+ 0.00127 [M+H-HCCl3]+ 0.00127

[M-H+2K]+ 0.05079 [M+H-NO2]+ 0.00571 [M+H-C8H10O2]+ 0.00254 [M+Li-C3H5NO2]+ 0.00127 [M+H-C2H3N3]+ 0.00127

[M+H-CO]+ 0.04635 [M+H-C6H13NO2]+ 0.00571 [M+H-C2Cl4]+ 0.00254 [M+Li-H3PO4]+ 0.00127 [M+H-C3H6O2]+ 0.00127

[M+H-CO2]+ 0.04318 [M-H-C3H5NO2]- 0.00508 [M-H-C7H5NO]- 0.00254 [M-2H+3Li-C15H31CO2H]+ 0.00127 [M+H-CH2Cl2O]+ 0.00127

[M+H-CH2O2]+ 0.03810 [M(81Br)-H]- 0.00508 [M+H-C5H11N]+ 0.00254 [M-2H+3Na-C3H5NO2]+ 0.00127 [M(356)+H-HCl]+ 0.00127

[M-H-NH3]- 0.03746 [M+H-HCO2H]+ 0.00508 [M+Ba-H]+ 0.00254 [M-2H+Na+Co]+ 0.00127 [M-C4H4O4S]+ 0.00127

[M.Cl]- 0.03556 [M-2H+Li]- 0.00444 [M+H-C14H25NO3]+ 0.00254 [M-2H+Li-C3H5NO2]- 0.00127 [M+H-C8H14O3]+ 0.00127

[M+Li]+ 0.03111 [M+H-CH4]+ 0.00444 [M+H-C6H5NO2S]+ 0.00254 [M-2H+Li-C16H30O]- 0.00127 [M+H-C2H4]+ 0.00127

Statistics: Adducts in NIST12 MS/MS DB (80,000 spectra)Most common adducts for LC-MS ([M+H]+ [M+Na]+ [M+NH4]+ [M+acetate]+)

…around 290 different adducts

ExPASy – the proteomic server

Different types of PTMs & their modification sites

Phosphorylation

Glycosylation

Acylation

Alkylation

Hydroxylation

Ser, Thr, Tyr

Asn, Ser, Thr

Asn, Gln, Lys

Lys, Arg

Pro, Lys

Process of post-translational modification

Cytosol

Endoplasmic reticulum

(ER)

P

P

GlcGlc

CH3CH3

Cleaved protein

Protein folding & PTMs

mRNARibosome

Protease

Removal of certain N- and

C-terminal residues

Translated Protein

Source: Modified from Biochemistry by A.L.Lehninger, 4th edition (ebook)

Increased complexity of proteome due to PTMs

A C G G U G C C G U G C A C GA C A C U A C G C A C U

Gene sequence

AsnGly

Ala

Val

His

Ala

LeuArg Thr

Expected protein structure

Actual protein structure

AsnGly

Ala

Val

His

AlaLeu

Arg Thr

PCH3

Glc

Phosphorylation reactions

Ser

R

CH2

CH

CH3

CH2

Thr

Tyr

ATP ADP

Kinase

Amino acid residue

Phosphorylated residue

OHC

NH3+

COO-

RH OC

NH3+

COO-

R PO43-H

Source: Modified from Biochemistry by A.L.Lehninger, 4th edition (ebook)

Glycosylation reactions

Ser/Thr

Asn

Glycosyl transferase

N-linked Glycosylation

O-linked Glycosylation

Glycosyl transferase

Sugar residues

N-linked amino acid

O-linked amino acid

CONH2C

NH3+

COO-

CH2HCONC

NH3+

COO-

CH2H

OHC

NH3+

COO-

RHOC

NH3+

COO-

RH

Source: Modified from Biochemistry by A.L.Lehninger, 4th edition (ebook)

Definitions of the components:Gel-based detection techniques for PTMs

1. Pro-Q-diamond: This fluorescent dye detects modified proteins that have been phosphorylated at serine, threonine or tyrosine residues. They are used with electrophoretic techniques and offer sensitivity down to few ng levels, depending upon the format in which they are used. This dye can also be combined with other staining procedures thereby allowing more than one detection protocol on a single gel.

a) Gel staining: The process by which the protein bands on an electrophoresis gel are stained by suitable dyes for visualization.

b) Gel scanning: The visualization of the stained protein bands on an electrophoresis gel by exciting it at a suitable maximum wavelength such that the dye absorbs the light and emits its own characteristic light at another emission wavelength.

2. Immunoblotting: This process, also known as Western blotting, is a commonly used analytical technique for detection of specific proteins in a given mixture by means of specific antibodies to the given target protein.

a) Electrophoresis: Electrophoresis is a gel-based analytical technique that is used for separation and visualization of biomolecules like DNA, RNA and proteins based on their fragment lengths or charge-to-mass ratios using an electric field. The protein mixture is first separated by means of a suitable electrophoresis technique such as SDS-PAGE or Two-dimensional Electrophoresis.

Definitions of the components:Gel-based detection techniques for PTMs

b) Blotting: The process by which the proteins separated on the electrophoresis gel are transferred on to another surface such as nitrocellulose by placing them in contact with each other.

c) Nitrocellulose sheet: A membrane or sheet made of nitrocellulose onto which the protein bands separated by electrophoresis are transferred for further probing and analysis.

d) Specific probe antibodies: Antibodies that are specific to a particular protein modification can be used as probes to detect those proteins containing that particular PTM. Protein phosphorylation is commonly detected using anti-phosphoserine, phosphothreonine or phosphotyrosine antibodies. Recently, specific motif antibodies have also been developed which detect a particular sequence of motif of the protein that contains a PTM.

e) Labeled secondary Abs: Antibodies labeled with a suitable fluorescent dye molecule are used to detect the interaction between the modified protein and its antibody by binding to another domain of the probe antibody.

Pro-Q-diamond staining

Completed 2-DE gel

Protein bands get fixed on gel and minimize diffusion.

Tubing connected & outlet opened

Dye stains the phosphorylated protein bands only.

Excess dye removed

Tray with fixing solution (methanol + acetic acid)Pro-Q-diamond stain

Washing solution (methanol + acetic

acid)

Gel scanning

Dec

rea

sing

m

ole

cula

r w

eig

ht

Decreasing pH

Gel scanner

Emission maxima – 580 nm

Phosphoprotein image

Stained gel

Gel removed from scanner

Dual staining with SYPRO-Ruby Red

SYPRO-Ruby red staining solution

Tubing connected & outlet opened

Dye stains all protein bands.Excess dye removed

Washing solution (methanol + acetic

acid)

Gel scanning

De

cre

asi

ng

mol

ecu

lar

we

ight

Decreasing pH

Gel scanner

Emission maxima – 610 nm

Total protein image

Flu

ore

sce

nce

Phosphoprotein image

Flu

ore

sce

nce

Total protein image by SYPRO-Ruby Red

A comparative profile between total protein image and phosphoprotein image enables detection of phosphorylated proteins.

Phosphoprotein image

Stained gel

Immunoblotting

Direction of migration

Anode

Cathode-

+

BufferAcrylamide gel

Sample loading

Protein mixture

SDS-PAGE 2-D Electrophoresis

Proteins focused on IPG strip

Direction of migration

Completed stained gels

Immunoblotting (this one for phosphorylated tyrosines!)

Completed gels

Nitrocellulose sheet or PVDFBlotting

Specific phospho-tyrosine

antibodies added

Detection using labeled secondary

antibodies

Proteins phosphorylated at

Tyr residues

Proteins phosphorylated at

Tyr residues

PHOSPHORYLATION

Phospho – ProteomicsWestern 2D gel , Ab specific to phospho-tyrosine

Phosphorylation and Mass Spec

Analysis of the entire complement of phosphorylated proteins in cells: “phosphoproteome” Qualitative and quantitative information regarding protein phosphorylation important in

many cellular processes signal transduction, gene regulation, cell cycle, apoptosis

Most common sites of phosphorylation: Ser, Thr, Tyr

MS can be used to detect and map locations for phosphorylation MW increase from addition of phosphate

group treatment with phosphatase allows

determination of number of phosphate groups

digestion and tandem MS allows for determination of phosphorylation sites

Enrichment strategies to analyze phosphoproteins/peptides

Chemical derivatization Introduce affinity tag to enrich for phosphorylated molecules

e.g., biotin binding to immobilized avidin/streptavidin

Enrichment strategies to analyze phosphoproteins/peptides

Oda et al., Nature Biotech. 2001, 19, 379 for analysis of pS and pT Remove Cys-reactivity by oxidation with performic acid Base hydrolysis induce ß-elimination of phosphate from pS/pT Addition of ethanedithiol allows coupling to biotin Avidin affinity chromatography to purify phosphoproteins

AND MORE~!

Enrichment strategies to analyze phosphoproteins/peptides

Phosphospecific antibodies Anti-pY quite successful Anti-pS and anti-pT not as successful, but may be used

(M. Grønborg, T. Z. Kristiansen, A. Stensballe, J. S. Andersen, O. Ohara, M. Mann, O. N. Jensen, and A. Pandey, “Approach for Identification of Serine/Threonine-phosphorylated Proteins by Enrichment with Phospho-specific Antibodies.” Mol. Cell. Proteomics 2002, 1:517–527.

Immobilized metal affinity chromatography (IMAC) Negatively charged phosphate groups bind to postively charged

metal ions (e.g., Fe3+, Ga3+) immobilized to a chromatographic support

Limitation: non-specific binding to acidic side chains (D, E) Derivatize all peptides by methyl esterification to reduce non-

specific binding by carboxylate groups. Ficarro et al., Nature Biotech. (2002), 20, 301.

Phosphoprotein and Sypro Ruby Stains with Laser Imaging

PeppermintStick phosphoprotein molecular weight standards (LifeTechnologies) separated on a 13% SDS polyacrylamide gel.

The gel was stained with Pro-Q Diamond phosphoprotein gel stain (blue) followed by SYPRO Ruby protein gel stain (red).

The digital images were pseudocolored

Phosphorylated

Beta-galactosidase

Bovine serum albumin (BSA)

Ovalbumin

Beta-casein

Avidin

lysozyme BAPTA

Phosphoprotein Stain

Visualization of total protein and phosphoproteins in a 2-D gel

Proteins from a Jurkat T-cell lymphoma line cell lysate separated by 2-D gel electrophoresis and stained with Pro-Q Diamond phosphoprotein gel stain (blue) followed by SYPRO Ruby protein gel stain (red). After each dye staining, the gel was imaged and the resulting composite image was digitally pseudocolored and overlaid.

T.H. Steinberg et al., Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology, Proteomics 2003, 3, 1128-1144

GLYCOSYLATION

Protein Glycosylation

• The most important and complex form of PTM

• Approx. 1% mammalian genes

• Early view about carbohydrates (non-specific, static structures) has been challenged

Ann. Rev. Biochem. 72(2003)643

Glycoprotein Gel Stain

CandyCane glycoprotein molecular weight standards (LifeTechnologies) containing alternating glycosylated and nonglycosylated proteins electrophoresed through a 13% polyacrylamide gel.

After separation, the gel was stained with SYPRO Ruby protein gel stain to detect all eight marker proteins (left). Subsequently, the gel was stained by the standard periodic acid–Schiff base (PAS) method in the Pro-Q Fuchsia Glycoprotein Gel Stain Kit to detect the glycoproteins alpha2-macroglobulin,

glucose oxidase, alpha1-glycoprotein and avidin.

Pro-Q™ Glycoprotein Stain (DDAO phosphate)Molecular Formula: C15H18Cl2N3O5P (MW 422.20)

Detection of glycoproteins and total protein on an SDS-polyacrylamide gel using the Pro-Q Fuchsia Glycoprotein Gel Stain Kit.

Protein Glycosylation Common in Eukaryotic Proteins

NITRATION

Nitro-Tyrosine Modification

Oxidative modification of amino acid side chains: methionine oxidation to the corresponding sulfone S-nitrosation or S-nitrosoglutationylation of cysteine residues Tyrosine modification to yield o,o’-dityrosine, 3-nitrotyrosine and 3-

chlorotyrosine. Tyrosine nitration is a well-established protein modification that occurs in disease states

associated with oxidative stress and increased nitric oxide synthase activity.

The combination of 2D-PAGE, western blotting, IMMUNOASSAY and mass spectrometry has been the more typical strategy to identify 3-nitrotyrosine-modified proteins.

Nitro-Tyrosine Modification

“Proteomic method identifies proteins nitrated in vivo during inflammatory challenge,” K. S. Aulak, M. Miyagi, L. Yan, K. A. West, D. Massillon, J. W. Crabb, and D. J. Stuehr, Proc. Natl. Acad. Sci. USA 2001; 98: 12056-12061.

Anti-nitrotyrosine immunopositive proteins in lung of rats induced with LPS.

WHAT WE DO AT OSU…

SERVICES at OSU Proteomics

• Protein Growth, Induction and Expression, Protein purification• Subcloning into recombinant cell lines, Plasmid design• DIGE • Develop novel protein protocols, individualized for experiment• Selective subfractionation, Salt fractionation, Enrichment, Solubility screening,

Inclusion body isolation• Western Blotting, Far Western Blotting, Immunoprecipitation and Co-

immunoprecipitation, Protein-Protein interaction studies• Classic chromatography:

Affinity –Tag purification, ionic exchange, HIC reverse phase, SEC gel chromatography 100,300, Immobilized metal affinity chromatography (IMAC), Heparin affinity: Protein A/G affinity column, ENDOTOXIN removal

• SDS-PAGE and DNA Electrophoresis, reduced and/or non-reduced• ProQ, LavaPurple, Sypro and other gel staining• Fluorescent and Bradford Protein Quantitation• Mass Spectrometry for protein identification

Just ask!

PTM identification!

Mass Spec and Proteomics and Protein Expression and Purification Facility

Biomedical Research Tower Room 250460 West 12th StreetColumbus, OhioLab: 614-247-8789

Arpad Somogyi, PhD – [email protected] L. James, PhD – [email protected]

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