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 posttranslational 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
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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 alpha2macroglobulin, glucose oxidase, alpha1-glycoprotein and
avidin.
Pro-Q™ Glycoprotein Stain (DDAO phosphate)
Molecular Formula: C15H18Cl2N3O5P (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 3chlorotyrosine.
 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 3nitrotyrosine-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.
Diesel Exhaust Particle-Induced Nitro-Tyrosine Modifications
RAW 264.7 macrophage exposed to DEP (Xiao, Loo, and Nel - UCLA)
anti-nitro-tyrosine
3.5
4.5
5.1
5.5
6.0
Sypro Ruby
7.0
8.4
9.53.5
4.5
5.1
5.5
6.0
7.0
8.4
9.5
kDa
116
HSP70
98
55
Naf-1
casein kinase II
enolase
37
30
MnSOD
20
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 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
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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
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 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.
Direct MS of phosphopeptides
bound to IMAC beads
 Raska et al., Anal.
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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
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Introduce affinity tag to enrich for
phosphorylated molecules
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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
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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)
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, betacasein, 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
6.0
7.0
98
55
37
30
Sypro Ruby
IEF
8.4 9.5
3.54.5 5.1 5.5
98
TNF convertase
MAGUK p55
PDI
Protein phosphatase 2A
JNK-1
p38 MAPK alpha
ERK-1
ERK-2
ErbB-2
TNF
HSP 27
20
Xiao, Loo, and Nel - UCLA
3
5
55
6 7
37
13
30
20
1
2
8
9
10
4
12
11
14
6.0
7.0
8.4 9.5
Mass Spectrometry and Quantitative
Measurements
H
equimolar mixture
of 2 peptides
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.
B
Rel. Abund.
A
Q
m/z
equimolar mixture
of 2 peptides
(M+2H)2+ : [12C]-ion
 = 0.036
[Val5]-Angiotensin II
Lys-des-Arg9-Bradykinin
1031.5188 (monoisotopic)
1031.5552 (monoisotopic)
516.725
m/z
516.828
Mass Spectrometry and Quantitative
Measurements
equimolar mixture
of 2 peptides
H
Q
E
13C 13C
Rel. Abund.
A
B
2D
2D
A
B
H
13C
Q
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 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 14Nminimal media, or stable isotope
amino acids (2D/13C/15N) or 15Nenriched 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 carboxyterminus of peptides
 Proteins from 2 different samples
are enzymatically digested in
normal water or H218O.
(Arg, Lys)
R1
R2
R3
R4
...NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOH
Trypsin /H218O
R1
R2
R3
R4
...NH-CH-CO-NH-CH-CO-18OH NH2-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
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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,
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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. 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