Transcript Slide 1
Mass Spectrometry for Protein Quantification and Identification of Posttranslational Modifications
Joseph A. Loo
Department of Biological Chemistry David Geffen School of Medicine Department of Chemistry and Biochemistry University of California Los Angeles, CA USA
Proteomics and posttranslational modifications
Eukaryotic cell.
Examples of protein properties are shown, including the interaction of proteins and protein modifications.
protein-ligand interactions protein complexes (machines) Patterson and Aebersold, Nature Genetics (supp.), 33, 311 (2003) post-translational modified proteins protein families (activity or structural)
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 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 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 Stain
Detection of glycoproteins and total protein on an SDS-polyacrylamide gel using the Pro-Q Fuchsia Glycoprotein Gel Stain Kit.
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 alpha 2 macroglobulin, glucose oxidase, alpha 1 -glycoprotein and avidin.
Pro Q™ Glycoprotein Stain (DDAO phosphate) Molecular Formula: C 15 H 18 Cl 2 N 3 O 5 P (MW 422.20)
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.
37 30 20 Diesel Exhaust Particle-Induced Nitro-Tyrosine Modifications RAW 264.7 macrophage exposed to DEP (Xiao, Loo, and Nel - UCLA) kDa 116 98
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Naf-1 casein kinase II enolase MnSOD trans. factor AP 2ß
G1
MAPK phosphatase 2
E2
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 tandem mass spectrometers precurson ion scanning using 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 (PO 3 ) Set Q3 to m/z 79: only species which fragment to give a fragment ion of 79 reach the detector and hence indicating phosphorylation detector Q1 Q2 collision cell Q3
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 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
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Immobilized metal affinity chromatography (IMAC) Negatively charged phosphate groups bind to postively charged metal ions (e.g., Fe 3+ , Ga 3+ ) 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 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 enrich 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)
Phospho
Phosphoprotein 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.
Phosphoprotein 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 ( 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
Global Analysis of Protein Phosphorylation RAW 264.7 exposed to DEP Pro-Q Diamond 3.54.5 5.1 5.5
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Mass Spectrometry and Quantitative Measurements
E equimolar mixture of 2 peptides A B H Q Mass 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.
m/z
equimolar mixture of 2 peptides [Val 5 ]-Angiotensin II 1031.5188 (monoisotopic) (M+2H) 2+ : [ 12 C]-ion
= 0.036
Lys-des-Arg 9 -Bradykinin 1031.5552 (monoisotopic) 516.725
m/z
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Mass Spectrometry and Quantitative Measurements
equimolar mixture of 2 peptides H Q E A B
2 D
A
2 D
B
13 C 13 C
H Q
13 C
E
m/z
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.
Methods coupling mass spectrometry and stable isotope tagging 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
MS/MS identifies the protein
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.
Stable Isotope Amino Acid or
15
N-
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 14 N minimal media, or stable isotope amino acids ( 2 D/ 13 C/ 15 N) or 15 N enriched media.
These mass tags are incorporated into proteins during translation.
Enzymatic Stable Isotope Coding of Proteomes
Enzymatic digestion in the presence of 18 O-water incorporates 18 O at the carboxy terminus of peptides Proteins from 2 different samples are enzymatically digested in normal water or H 2 18 O.
R 1 (Arg, Lys) R 2 R 3 R 4
...NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOH
R 1 R 2 Trypsin /H 2
18 O
R 3 R 4
...NH-CH-CO-NH-CH-CO 18 O H NH 2 -CH-CO-NH-CH-COOH
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 Analysis Reiner Westermeier, Tom Naven Wiley-VCH, 2002
PART I: PROTEOMICS TECHNOLOGY
Introduction Expression Proteomics Two-dimensional Electrophoresis Spot Handling Mass Spectrometry Protein Identification by Database Searching Methods of Proteomics
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
Proteins and Proteomics: A Laboratory Manual Richard J. Simpson Cold Spring Harbor Laboratory (2002)
Chapter 1.
Introduction to Proteomics
Chapter 2.
One –dimensional Polyacrylamide Gel Electrophoresis
Chapter 3.
Chapter 4.
Preparing Cellular and Subcellular Extracts Preparative Two –dimensional Gel Electrophoresis with
Chapter 5.
Chapter 6.
Chapter 7.
Immobilized pH Gradients Reversed –phase High–performance Liquid Chromatography Amino – and Carboxy– terminal Sequence Analysis 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