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Mass Spectrometry for Protein Quantification and Identification of Posttranslational Modifications

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Title: Mass Spectrometry for Protein Quantification and Identification of Posttranslational Modifications


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
2
Proteomics and posttranslational modifications
Eukaryotic cell. Examples of protein properties
are shown, including the interaction of proteins
and protein modifications.
Patterson and Aebersold, Nature Genetics (supp.),
33, 311 (2003)
3
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

4
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.

5
  • 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)

6
Proteomic analysis of PTMs
Mann and Jensen, Nature Biotech. 21, 255 (2003)
7
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 acidSchiff 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)
8
Nitro-Tyrosine Modification
  • Oxidative modification of amino acid side chains
    include methionine oxidation to the corresponding
    sulfone, S-nitrosation or S-nitrosoglutationylatio
    n 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 NO2
  • 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.

9
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.
10
Diesel Exhaust Particle-Induced Nitro-Tyrosine
Modifications
RAW 264.7 macrophage exposed to DEP (Xiao, Loo,
and Nel - UCLA)
Sypro Ruby
anti-nitro-tyrosine
HSP70
Naf-1
enolase
casein kinase II
MnSOD
MAPK phosphatase 2
trans. factor AP-2ß
E2
G1
11
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

12
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

13
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

detector
Q1
Q2 collision cell
Q3
14
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

15
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,
    1517527.
  • 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.

16
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)

17
  • 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
18
Enrichment strategies to analyze
phosphoproteins/peptides
  • Chemical derivatization
  • Introduce affinity tag to enrich for
    phosphorylated molecules
  • e.g., biotin binding to immobilized
    avidin/streptavidin

19
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

20
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

21
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)

22
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.
Phospho
23
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
24
Global Analysis of Protein Phosphorylation
RAW 264.7 exposed to DEP
Pro-Q Diamond
Sypro Ruby
IEF
98
98
3
TNF? convertase MAGUK p55 PDI Protein phosphatase
2A JNK-1 p38 MAPK alpha ERK-1 ERK-2 ErbB-2 TNF HSP
27
5
55
55
7
6
1
2
37
37
8
13
9
4
10
30
30
12
11
14
20
20
Xiao, Loo, and Nel - UCLA
25
Mass Spectrometry and Quantitative Measurements
equimolar mixture of 2 peptides
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 of 2 peptides
? 0.036
(M2H)2 12C-ion
Val5-Angiotensin II 1031.5188 (monoisotopic)
Lys-des-Arg9-Bradykinin 1031.5552 (monoisotopic)
m/z
26
Mass Spectrometry and Quantitative Measurements
equimolar mixture of 2 peptides
Rel. Abund.
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.
27
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).

28
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.

29
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.

30
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 H218O.

31
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

32
Identification of Low Abundance Proteins
GenWay Biotech
33
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.

34
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 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
35
Proteins and Proteomics A Laboratory
ManualRichard J. Simpson Cold Spring Harbor
Laboratory (2002)
Chapter 1. Introduction to Proteomics Chapter 2.
Onedimensional Polyacrylamide Gel
Electrophoresis Chapter 3. Preparing Cellular
and Subcellular Extracts Chapter 4. Preparative
Twodimensional Gel Electrophoresis with
Immobilized pH Gradients Chapter 5.
Reversedphase Highperformance Liquid
Chromatography Chapter 6. Amino and Carboxy
terminal Sequence Analysis Chapter 7. Peptide
Mapping and Sequence Analysis of Gelresolved
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 Proteins
Structure, Functions, and Interactions
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