Mass Spectrometry for Protein Quantification and Identification of Posttranslational

ModificationsJoseph A. LooJoseph A. Loo

Department of Biological ChemistryDepartment of Biological ChemistryDavid Geffen School of MedicineDavid Geffen School of Medicine

Department of Chemistry and Department of Chemistry and BiochemistryBiochemistry

University of CaliforniaUniversity of CaliforniaLos Angeles, CA USALos Angeles, CA USA

Proteomics and posttranslational modifications

Patterson and Aebersold, Nature Genetics (supp.), 33, 311 (2003)

protein-ligandprotein-ligandinteractionsinteractions

protein-ligandprotein-ligandinteractionsinteractions

proteinproteincomplexescomplexes(machines)(machines)

proteinproteincomplexescomplexes(machines)(machines)

protein familiesprotein families(activity or structural)(activity or structural)

protein familiesprotein families(activity or structural)(activity or structural)

post-translationalpost-translationalmodified proteinsmodified proteins

post-translationalpost-translationalmodified proteinsmodified proteins

Eukaryotic cell.Examples of protein

properties are shown, including the interaction of proteins

and protein modifications.

Proteomic Analysis of Post-translational Modifications

Post-translational modifications (PTMs) Covalent processing events that change the properties

of a protein proteolytic cleavage addition of a modifying group to one or more amino

acids Determine its activity state, localization, turnover,

interactions with other proteins Mass spectrometry and other biophysical methods can

be used to determine and localize potential PTMs However, PTMs are still challenging aspects of

proteomics with current methodologies

Complexity of the ProteomeComplexity of the Proteome

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

Complexity of the human proteome is far beyond the more than 30,000 human genes.

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 advance beyond identifying proteins to Proteomic approaches that advance beyond identifying proteins to elucidating their post-translational modifications are needed.elucidating their post-translational modifications are needed.

Use MS to determine PTM of isolated protein

Enzymatic or chemical degradation of modified protein

HPLC separation of peptides

MALDI and/or ESI used to identify PTM

MS/MS used to determine location of PTM(s)

Proteomic analysis of PTMs

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

Glycoprotein Gel StainGlycoprotein Gel Stain

CandyCane glycoprotein molecular weight standards containing alternating glycosylated and nonglycosylated proteins were 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.

Nitro-Tyrosine Modification

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

Nitric oxide (NO) synthases provide the biological precursor for nitrating agents that perform this modification in vivo. NO can form nitrating agents in a number of ways including reacting with superoxide to make peroxynitrite (HOONO) and through enzymatic oxidation of nitrite to generate NO·

2

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, 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.

116

98

55

37

30

20

kDa

3.5 9.54.5 5.1 5.5 6.0 7.0 8.4 3.5 9.54.5 5.1 5.5 6.0 7.0 8.4

MAPK phosphatase 2MAPK phosphatase 2E2

G1

enolaseenolasecasein kinase IIcasein kinase II

HSP70HSP70

Naf-1Naf-1

Diesel Exhaust Particle-Induced Nitro-Tyrosine ModificationsDiesel Exhaust Particle-Induced Nitro-Tyrosine Modifications

RAW 264.7 macrophage exposed to DEP (Xiao, Loo, and Nel - UCLA)

Sypro Rubyanti-nitro-tyrosine

trans. factor AP-2ßtrans. factor AP-2ß

MnSODMnSOD

Phosphorylation

Analysis of the entire complement of phosphorylated proteins in cells: “phosphoproteome”

Qualitative and quantitative information regarding protein phosphorylation important

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

MS/MS and Phosphorylation

Detection of phosphopeptides in complex mixtures can be facilitated by neutral loss and precurson ion scanning using tandem mass spectrometers

Allow selective visualization of peptides containing phosphorylated residues

Most commonly performed with triple quadrupole mass spectrometers

precursor ion transmission

collision cell (chamber)

mass analysis of product ions

MS/MS and Phosphorylation

Precursor ion scan Q1 is set to allow all the components of the mixture to enter the

collision cell and undergo CAD Q3 is fixed at a specific mass value, so that only analytes which

fragment to give a fragment ion of this specific mass will be detected

Phospho-peptide fragments by CAD to give an ion at m/z 79 (PO3) Set Q3 to m/z 79: only species which fragment to give a fragment

ion of 79 reach the detector and hence indicating phosphorylation

Q1 Q2collision cell

Q3

detector

MS/MS and Phosphorylation

Neutral loss scan Q1 and Q3 are scanned synchronously but with a

specific m/z offset The entire mixture is allowed to enter the collision cell,

but only those species which fragment to yield a fragment with the same mass as the offset will be observed at the detector

pSer and pThr peptides readily lose phosphoric acid during CAD (98 Da)

For 2+ ion set offset at 49 Any species which loses 49 from a doubly charged ion

would be observed at the detector and be indicative of phosphorylation

Enrichment strategies to analyze phosphoproteins/peptides

Phosphospecific antibodiesPhosphospecific 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)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.

Direct MS of phosphopeptides bound to IMAC beads

Raska et al., Anal. Chem. 2002, 74, 3429

IMAC beads placed directly on MALDI target

Matrix solution spotted onto target

MALDI-MS of peptides bound to IMAC bead

MALDI-MS/MS (*) to identify phosphorylation site(s)

MALDI-MS spectrum obtained from peptide bound to IMAC beads applied directly to MALDI target

MALDI-MS/MS (Q-TOF) to locate phosphorylation site

Sample enrichment with minimal sample handling

contains phosphorylated

residue

Enrichment strategies to analyze phosphoproteins/peptides

Chemical derivatizationChemical 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

Enrichment strategies to analyze phosphoproteins/peptides

Zhou et al., Nature Biotech. 2001, 19, 375 Reduce and alkylate Cys-residues to eliminate their

reactivity Protect amino groups with t-butyl-dicarbonate (tBoc) Phosphoramidate adducts at

phosphorylated residues are formed by carbodiimide condensation with cystamine

Free sulfhydryls are covalently captured onto glass beads coupled to iodoacetic acid

Elute with trifluoroacetic acid

Chemical derivatization to Chemical derivatization to enrich for phosphoproteinsenrich for phosphoproteins

Developed because other methods based on affinity/adsorption (e.g., IMAC) displayed some non-specific binding

Chemical derivatization methods may be overly complex to be used routinely

Sensitivity may not be sufficient for some experiments (low pmol)

Phosphoprotein StainPhosphoprotein Stain

PeppermintStick phosphoprotein molecular weight standards separated on a 13% SDS polyacrylamide gel. The markers contain (from largest to smallest) beta-galactosidase, bovine serum albumin (BSA), ovalbumin, beta-casein, avidin and lysozyme. Ovalbumin and beta-casein are phosphorylated. 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.

Phospho

Phosphoprotein StainPhosphoprotein Stain

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

Proteins from a Jurkat T-cell lymphoma line cell lysate were separated by 2-D gel electrophoresis and stained with Pro-Q Diamond phosphoprotein gel stain (blueblue) followed by SYPRO Ruby protein gel stain (redred). 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

RAW 264.7 exposed to DEP

Global Analysis of Protein PhosphorylationGlobal Analysis of Protein Phosphorylation

Pro-Q DiamondPro-Q Diamond Sypro RubySypro Ruby

Xiao, Loo, and Nel - UCLA

IEF

9.53.54.5 5.1 5.5 6.0 7.0 8.4

53

4

12

6 7

20

30

37

98

55

9.53.54.5 5.1 5.5 6.0 7.0 8.4

30

37

98

55

20

89

10

1112

13

14

TNFTNF convertase convertaseMAGUK p55MAGUK p55

PDIPDIProtein phosphatase 2AProtein phosphatase 2A

JNK-1JNK-1p38 MAPK alphap38 MAPK alpha

ERK-1ERK-1ERK-2ERK-2ErbB-2ErbB-2

TNFTNFHSP 27HSP 27

AA B

m/z

Re

l. A

bun

d.

QQH EEMass spectrometry is inherently not a quantitative technique. The intensity of a peptide ion signal does not accurately reflect the amount of peptide in the sample.

equimolar mixture equimolar mixture of 2 peptidesof 2 peptides

516.725 516.828m/z

(M+2H)2+ : [12C]-ion

[Val5]-Angiotensin II1031.5188 (monoisotopic)

Lys-des-Arg9-Bradykinin1031.5552 (monoisotopic)

= 0.036= 0.036equimolar mixture equimolar mixture of 2 peptidesof 2 peptides

Mass Spectrometry and Quantitative Measurements

AA B

m/z

Re

l. A

bun

d.

QQH EE

Two peptides of identical chemical structure that differ in mass because they differ in isotopic composition are expected to generate identical specific signals in a mass spectrometer.

equimolar mixture equimolar mixture of 2 peptidesof 2 peptides

Mass Spectrometry and Quantitative Measurements

QQH EE

13C13C13C

AA B

2D 2D

Methods coupling mass spectrometry and stable isotope tagging Methods coupling mass spectrometry and stable isotope tagging have been developed for quantitative proteomics.have been developed for quantitative proteomics.

ICAT: Isotope-Coded Affinity Tag

Alkylating group covalently attaches the reagent to reduces Cys-residues. A polyether mass-encoded linker contains 8 hydrogens (d0) or 8 deuteriums

(d8) that represents the isotope dilution. A biotin affinity tag is used to selectively isolate tagged peptides (by avidin

purification).

ICAT: Isotope-Coded Affinity Tag

The Cys-residues in sample 1 is labeled with d0-ICAT and sample 2 is labeled with d8-ICAT. The combined samples are digested, and the biotinylated ICAT-labeled peptides are enriched by avidin

affinity chromatography and analyzed by LC-MS/MS. Each Cys-peptide appears as a pair of signals differing by the mass differential encoded in the tag. The

ratio of the signal intensities indicates the abundance ratio of the protein from which the peptide originates.

MS/MS identifies the protein

Stable Isotope Amino Acid or 15N- in vivo Labeling

Metabolic stable isotope coding of proteomes

An equivalent number of cells from 2 distinct cultures are grown on media supplemented with either normal amino acids or 14N-minimal media, or stable isotope amino acids (2D/13C/15N) or 15N-enriched media.

These mass tags are incorporated into proteins during translation.

Enzymatic Stable Isotope Coding of Proteomes

Enzymatic digestion in the presence of 18O-water incorporates 18O at the carboxy-terminus of peptides

Proteins from 2 different samples are enzymatically digested in normal water or H2

18O.

RR33 RR44

NH2-CH-CO-NH-CH-COOH...NH-CH-CO-NH-CH-CO-...NH-CH-CO-NH-CH-CO-1818OOHH

RR11 RR22

...NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOH...NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOH

RR11 RR22 RR33 RR44

Trypsin /HTrypsin /H221818OO

(Arg, Lys)(Arg, Lys)

C-terminal peptide

Identification of Low Abundance Proteins

The identification of low abundance proteins in the presence of high abundance proteins is problematic (e.g., “needle in a haystack”)

Pre-fractionation of complex protein mixtures can alleviate some difficulties gel electrophoresis, chromatography,

etc Removal of known high abundance

proteins allows less abundant species to be visualized and detected

Identification of Low Abundance Proteins

GenWay Biotech

Additional Readings

R. Aebersold and M. Mann, Mass spectrometry-based proteomics, Nature (2003), 422, 198-207.

M. B. Goshe and R. D. Smith, “Stable isotope-coded proteomic mass spectrometry.” Curr. Opin. Biotechnol. 2003; 14: 101-109.

W. A. Tao and R. Aebersold, “Advances in quantitative proteomics via stable isotope tagging and mass spectrometry.” Curr. Opin. Biotechnol. 2003; 14: 110-118.

S. D. Patterson and R. Aebersold, “Proteomics: the first decade and beyond.” Nature Genetics 2003; 33 (suppl.): 311-323.

M. Mann and O. N. Jensen, “Proteomic analysis of post-translational modification.” Nature Biotech. 2003; 21: 255-261.

D. T. McLachlin and B. T. Chait, “Analysis of phosphorylated proteins and peptides by MS.” Curr. Opin. Chem. Biol. 2001; 5: 591-602.

S. Gygi et al., “Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.” Nature Biotech. 1999; 17: 994-999.

Proteomics in Practice: A Laboratory Manual of Proteome AnalysisReiner Westermeier, Tom NavenWiley-VCH, 2002

PART II: COURSE MANUAL Step 1: Sample Preparation Step 2: Isoelectric Focusing Step 3: SDS Polyacrylamide Gel Electrophoresis Step 4: Staining of the Gels Step 5: Scanning of Gels and Image Analysis Step 6: 2D DIGE Step 7: Spot Excision Step 8: Sample Destaining Step 9: In-gel Digestion Step 10: Microscale Purification Step 11: Chemical Derivatisation of the Peptide Digest Step 12: MS Analysis Step 13: Calibration of the MALDI-ToF MS Step 14: Preparing for a Database Search Step 15: PMF Database Search Unsuccessful

PART I: PROTEOMICS TECHNOLOGY Introduction Expression Proteomics Two-dimensional Electrophoresis Spot Handling Mass Spectrometry Protein Identification by Database Searching Methods of Proteomics

Proteins and Proteomics: A Laboratory ManualRichard J. SimpsonCold Spring Harbor Laboratory (2002)

Chapter 1. Introduction to Proteomics Chapter 2. One–dimensional Polyacrylamide Gel Electrophoresis Chapter 3. Preparing Cellular and Subcellular Extracts Chapter 4. Preparative Two–dimensional Gel Electrophoresis with

Immobilized pH Gradients Chapter 5. Reversed–phase High–performance Liquid Chromatography Chapter 6. Amino– and Carboxy– terminal Sequence Analysis Chapter 7. Peptide Mapping and Sequence Analysis of Gel–resolved Proteins Chapter 8. The Use of Mass Spectrometry in Proteomics Chapter 9. Proteomic Methods for Phosphorylation Site Mapping Chapter 10. Characterization of Protein Complexes Chapter 11. Making Sense of Proteomics: Using Bioinformatics to Discover a

Protein’s Structure, Functions, and Interactions