MALDI TOF protein modification
Mass Spectrometry for Protein Quantification and Identification of Posttranslational Modifications
Joseph A. Loo Joseph A. Loo Department of Biological Chemistry Department of Biological Chemistry David Geffen School of Medicine David Geffen School of Medicine Department of Chemistry and Biochemistry Department of Chemistry and Biochemistry
University of California University of California Los Angeles, CA USA Los Angeles, CA USA
Proteomics and posttranslational modifications
Patterson and protein-ligand protein-ligand interactions interactions protein-ligand protein-ligand interactions interactions protein protein complexes complexes(machines) (machines) protein protein complexes complexes
(machines) (machines) 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
Complexity of the Proteome 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
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
Glycoprotein Gel Stain 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 2 - Glycoprotein Gel Stain Kit to detect the glycoproteins alpha macroglobulin, glucose oxidase, alpha -glycoprotein and 1 avidin.
Pro-Q™ Glycoprotein Stain (DDAO phosphate) Molecular Formula: C H Cl N O P (MW 422.20) 15 18 2 3 5
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 withsuperoxide 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
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.
Diesel Exhaust Particle-Induced Nitro-Tyrosine Modifications
Diesel Exhaust Particle-Induced Nitro-Tyrosine Modifications
RAW 264.7 macrophage exposed to DEP (Xiao, Loo, and Nel - UCLA)
anti-nitro-tyrosine
Sypro Ruby
3.5
4.5
5.1
5.5
6.0
7.0
8.4
9.5 3.5 4.5 5.1
5.5
6.0
7.0
8.4
9.5 116 kDa HSP70 98 HSP70
Naf-1 55 Naf-1 enolase enolase casein kinase II 37 casein kinase II
MnSOD
30 MnSOD20
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
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
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
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 Enrichment strategies to analyze phosphoproteins/peptides
Phosphospecific antibodies Phosphospecific antibodies
Anti-pY quite successful
(M.
Anti-pS and anti-pT not as successful, but may be used 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 3+ 3+ metal ions (e.g., Fe , Ga ) 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.
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 Enrichment strategies to analyze phosphoproteins/peptides
Chemical derivatization 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
Chemical derivatization to
enrich for phosphoproteins
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
Phosphoprotein Stain
Phosphoprotein Stain
Phospho
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 Phosphoprotein Stain 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
blue )
phosphoprotein gel stain (blue followed by SYPRO Ruby protein gel
red ). After each dye staining,
stain (red the gel was imaged and the resulting composite image was digitally pseudocolored and overlaid. RAW 264.7 exposed to DEP
Global Analysis of Protein Phosphorylation Global Analysis of Protein Phosphorylation
Pro-Q Diamond Pro-Q Diamond
Sypro Ruby Sypro Ruby
IEF 9.5 3.54.5 5.1 5.5 6.0 7.0 8.4
5 3
4
1 2 6 7
20 30 37 98 55 9.5 3.54.5 5.1 5.5 6.0 7.0 8.4
30 37 98 55
20 8 9 10 11 12 13 14 TNF TNF convertase convertase
MAGUK p55 MAGUK p55
PDI PDI Protein phosphatase 2A Protein phosphatase 2A
JNK-1 JNK-1 p38 MAPK alpha p38 MAPK alpha ERK-1
ERK-1 ERK-2
ERK-2 ErbB-2 ErbB-2 TNF TNF
HSP 27 HSP 27 A
A
B
m/z R e l.
A b un d.
Q Q H E
E 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. equimolar mixture equimolar mixture of 2 peptides of 2 peptides
(M+2H) 2+ : [ 12 C]-ion [Val 5 ]-Angiotensin II Lys-des-Arg 9 -Bradykinin
= 0.036 = 0.036 equimolar mixture equimolar mixture of 2 peptides of 2 peptides
Mass Spectrometry and Quantitative
MeasurementsMass Spectrometry and Quantitative Measurements equimolar mixture equimolar mixture
E
E H
Q
Q
of 2 peptides of 2 peptides 13 13 13 2 2 C C C
D D
A BA
E
E H
Q
Q d.
A n A B bu A l. e R 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.
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.
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
15 Stable Isotope Amino Acid or 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 14 either normal amino acids or N- minimal media, or stable isotope 2 13 15 15 amino acids ( D/ C/ N) or N- enriched media.
These mass tags are incorporated into proteins during Enzymatic Stable Isotope Coding of Proteomes
18
Enzymatic digestion in the presence of O- 18 water incorporates O at the carboxy-terminus of peptides
Proteins from 2 different samples are enzymatically digested in normal water or H 218 O.
(Arg, Lys) (Arg, Lys) R R R R R R R R 1 1 2 2 3 3 4 4
...NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOH ...NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOH 18 18 Trypsin /H Trypsin /H O O 2 2
Identification of Low Abundance Proteins
The identification of low abundance
proteins in the presence of highabundance 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 Identification of Low Abundance Proteins
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.
Proteomics in Practice: A Laboratory Manual of Proteome Analysis Reiner Westermeier, Tom Naven Wiley-VCH, 2002
PART II: COURSE MANUAL Step 1: Sample Preparation Step 2: Isoelectric Focusing Step 3: SDS Polyacrylamide Gel Electrophoresis PART I: PROTEOMICS Step 4: Staining of the Gels TECHNOLOGY Step 5: Scanning of Gels and Image Analysis Introduction
Step 6: 2D DIGE Expression Proteomics Step 7: Spot Excision Two-dimensional Electrophoresis Step 8: Sample Destaining Spot Handling Step 9: In-gel Digestion Mass Spectrometry Step 10: Microscale Purification Protein Identification by Database Step 11: Chemical Derivatisation of the Peptide Digest Searching Step 12: MS Analysis Methods of Proteomics 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. 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