Transcript post-translational modifications.

Quantitative proteomics
Peking Union Medical College
Chinese Academy of Medical Sciences
Wei Sun
[email protected]
Content
 1.Introduction
 2.
MS-based methods
 3.
Gel-based methods
Petterson SD, et al. Nat Genetics, 2003, 33, 311-23
Petterson SD, et al. Nat Genetics, 2003, 33, 311-23
Expression Proteomics

Expression proteomics:quality and quantity
of the proteins expressed of the cell.
 Technology:
1.Isolation: SDS-PAGE gel, HPLC (high
performance liquid chromatography), CE
(capillary electrophoresis)
2.Identification: mass spectrometry
3.Quantitation: ICAT, DIGE
Function Proteomics

Function proteomics: the function of the
proteins, mainly proteins interaction.
 Technology:
1.yeast two hybrid
2.phage display
3.TAP(tandem affinity purification)
Petterson SD, et al. Nat Genetics, 2003, 33, 311-23
Introduction

1. Quantitation proteomics:

The global analysis of protein expression,
a complementary method to study steadystate gene expression and perturbationinduced changes.
Gygi,S.P,et al. Nat Biotech, 1999, 17, 994-9
The measurement of the celluar response
to external perturbations at the mRNA and
protein level are complementary
6200-997-289
Ideker T, et al. Science,2001,292,929-934
Applications of Quantitative Proteomics

Indentify differenial expressed protein in
different states
 Detect alternation in protein post-translational
modification
 Protein complex characterization
 Protein-protein interactions
Quantitative proteomics analysis of yeast
grown in ethanol versus galactose
Gygi et al. Nature Biotech, 1999, 17:994-9
Gygi et al. Nature Biotech, 1999, 17:994-9
Quantitative proteomic analysis of Myc oncoprotein
function
Shiio Y, EMBO, 2002,21,5088-96
Application

Characterization of yeast RNA polymerase II
transcription preinitiation complex
 Microsomal proteins: pharmacologically
induced differentiation in human myeloid
leukemia
 Protein expression between control and
camptothecin-treated mouse cortical neurons
MS-based methods

1.Separation: 2D-LC/MS/MS (SCX-RP)

2. Identification: mass spectrometry and
database searching algorithm

3. Label: chemical probes
MS-based methods
Yates JR, et al. Nat Biotech, 2001, 19,242-7
MS-based quantitation
Chemical probes

Which isotope should be used?

What is the purity of the labeling reagent?

How many isotope labeled residues will be
present in each peptide?

Will the labeling tag remain intact during
peptide ion fragmentation?
Isotope-coded affinity tags (ICAT)
Gygi,S.P.,et al. Nat Biotech, 1999, 17, 994-9
Advantages

1. The method is compatible with any amount of
protein harvested from bodily fluids, cells or tissues
under any growth conditions.
 2. The alkylation reaction is highly specific and occurs
in the presence of salts, detergents, and stabilizers (e.g.
SDS, urea, guanidine-HCl).
 3. The complexity of the peptide mixture is reduced
by isolating only cysteine-containing peptides.
 4. The ICAT strategy permits almost any type of
biochemical, immunological, or physical
fractionization, which makes it compatible with the
analysis of low- abundance proteins.
Gygi,S.P.,et al. Nat Biotech, 1999, 17, 994-9
Disadvantages

1. The size of the ICAT label (~500 Da) is a large
modification that remains on each peptide throughout the
MS analysis. This can complicate the database searching
algorithms, especially for small peptides (<7 amino acids).
 2. The elution separation of light and heavy isotopes.
 3. The method fails for proteins that contain no cysteines.
Only a small percentage of proteins are cysteine-free (8%
in yeast).
 4. The avidin columns used for the affinity separation of the
biotin labeled peptides can present challenges, including
nonspecific binding, irreversible binding and low capacity.
 5. Label efficiency is relative low. (80%)
 6. The cysteine-based ICAT tags would not yield
information on changes in the proteome based on posttranslational modifications.
Gygi,S.P.,et al. Nat Biotech, 1999, 17, 994-9
Solution(1)






1. The size of the ICAT label (~500 Da) is a large
modification that remains on each peptide throughout the
MS analysis. This can complicate the database searching
algorithms, especially for small peptides (<7 amino acids).
2. The elution separation of light and heavy isotopes.
3. The method fails for proteins that contain no cysteines.
Only a small percentage of proteins are cysteine-free (8%
in yeast).
4. The avidin columns used for the affinity separation of the
biotin labeled peptides can present challenges, including
nonspecific binding, irreversible binding and low capacity.
5. Label efficiency is relative low. (80%)
6. The cysteine-based ICAT tags would not yield
information on changes in the proteome based on posttranslational modifications.
Solid-phase isotope tagging
Aebersold R, et al. Nat Biotech, 2002, 19,512-5
Aebersold R, et al. Nat Biotech, 2002, 19,512-5
Acid-labile isotope coded
extractants (ALICE)
Wang JH, et al. Anal Chem, 2002,74,4969-79
Wang JH, et al. Anal Chem, 2002,74,4969-79
Solution(2)






1. The size of the ICAT label (~500 Da) is a large
modification that remains on each peptide throughout the
MS analysis. This can complicate the database searching
algorithms, especially for small peptides (<7 amino acids).
2. The elution separation of light and heavy isotopes.
3. The method fails for proteins that contain no cysteines.
Only a small percentage of proteins are cysteine-free (8%
in yeast).
4. The avidin columns used for the affinity separation of the
biotin labeled peptides can present challenges, including
nonspecific binding, irreversible binding and low capacity.
5. Label efficiency is relative low. (80%)
6. The cysteine-based ICAT tags would not yield
information on changes in the proteome based on posttranslational modifications.
13C-Isotope-coded Affinity Tag
Burlingame AL, et al. MCP,
2003,2, 299-314
Regnier, FE, et al. J Proteome Res,
2002, 1, 139-47
13C-Isotope-coded Affinity Tag
Solution(3)






1. The size of the ICAT label (~500 Da) is a large
modification that remains on each peptide throughout the
MS analysis. This can complicate the database searching
algorithms, especially for small peptides (<7 amino acids).
2. The elution separation of light and heavy isotopes.
3. The method fails for proteins that contain no cysteines.
Only a small percentage of proteins are cysteine-free (8%
in yeast).
4. The avidin columns used for the affinity separation of the
biotin labeled peptides can present challenges, including
nonspecific binding, irreversible binding and low capacity.
5. Label efficiency is relative low. (80%)
6. The cysteine-based ICAT tags would not yield
information on changes in the proteome based on posttranslational modifications.
Chemical probes
Aebersold R, et al. Curr Opin Chem Bio, 2004, 8, 66-75
N-terminus
Liebler DC, et al. J Proteome Res, 2003, 2, 265-72
James P, et al.Anal Chem, 2000, 72, 4047-57
C-terminus
Fenselau C, et al. Anal Chem, 2001, 73, 2836-42
Tryptophan
Nishimura O, et al.Rapid Commun Mass Spectrom, 2003, 17, 1642-50
Mass-coded abundance
tagging (MCAT)
Emili A, et al.Nat Biotech,2002, 20, 163-70
Reilly JP, et al. Rapid Commun Mass Spectrom, 2000, 14, 2147-53
Emili A, et al.Nat Biotech,2002, 20, 163-70
Element-Coded Affinity Tags (ECAT)
Whetstone PA, et al.Bioconjugate Chem, 2004,15, 3-6
Solution(4)

1. The size of the ICAT label (~500 Da) is a large
modification that remains on each peptide throughout the
MS analysis. This can complicate the database searching
algorithms, especially for small peptides (<7 amino acids).
 2. The elution separation of light and heavy isotopes.
 3. The method fails for proteins that contain no cysteines.
Only a small percentage of proteins are cysteine-free (8%
in yeast).
 4. The avidin columns used for the affinity separation of
the biotin labeled peptides can present challenges,
including nonspecific binding, irreversible binding and low
capacity.
 5. Label efficiency is relative low. (80%)
 6. The cysteine-based ICAT tags would not yield
information on changes in the proteome based on posttranslational modifications.
Cell Culture
Fu EW, et al. Rapid Commun Mass Spectrom, 2002, 16, 1389-97
Stable Isotope Labeling by Amino
Acids in Cell Culture (SILAC)
Mann M, et al. MCP
2002, 1,376-86
Gygi SP, et al.MCP,
2004, in press.
Disadvantages

1. The method does not allow for the analysis of
protein directly from tissue.
 2. The stable-isotope- enriched media might themselves
affect microbial growth and protein production.
 3. Stable- isotope-enriched media are costly, and for
culturing cells from higher organisms they may be
impossible to obtain.
 4. The increase in nominal mass due to stable-isotope
incorporation is not known until the sequence is
determined, which can greatly confound databasesearching programs and prevent protein identification
prior to quantification.
Gygi SP, et al. Curr Opin Biotech. 2000,11,396-401
Solution






1. The size of the ICAT label (~500 Da) is a large
modification that remains on each peptide throughout the
MS analysis. This can complicate the database searching
algorithms, especially for small peptides (<7 amino acids).
2. The elution separation of light and heavy isotopes.
3. The method fails for proteins that contain no cysteines.
Only a small percentage of proteins are cysteine-free (8%
in yeast).
4. The avidin columns used for the affinity separation of
the biotin labeled peptides can present challenges,
including nonspecific binding, irreversible binding and low
capacity.
5. Label efficiency is relative low. (80%)
6. The cysteine-based ICAT tags would not yield
information on changes in the proteome based on posttranslational modifications.
Yates JR, et al. Anal Chem, 2002, 74, 1650-7
Julka S, et al. J Proteome Res, 2003, 3, 350-63
Gel-based methods
Aebersold R, et al. MCP, 2002, 1,19-29
Hamdan M, et al. Rapid Commun Mass Spectrom, 2002, 16, 1692-8
Hamdan M, et al. Rapid Commun Mass Spectrom, 2002, 16, 1692-8
Sechi S, et al. Rapid Commun Mass Spectrom, 2002, 16, 1416-24
Chemically-coded affinity tag (CCAT)
Niehaus K, et al. 2003, J Biotech, 106, 287-300
Niehaus K, et al. 2003, J Biotech, 106, 287-300
Differential In-gel Electrophoresis (DIGE)
Unlu, et al. Electrophoresis 18, 2071–207
Advantages

1. The control and experimental samples are
mixed in the same gel, no separate standard
maps must be created for the controls and treated
ones.
 2. Matching is automatic and straightforward
and a single gel could suffice for full
quantitative analysis.
Righetti G, et al. Mass Spectrom Rev, 2002, 21, 287-302
Disadvantages

1. In order to maintain solubility of the
labeled proteins during electrophoresis, one
must fluorescently derivatize the sample such
that only ~1–2% of the lysine residues of the
proteins are modified. Higher labeling
stoichiometries severely compromise the
solubility of the proteins and greatly decrease
the number of proteins detected. So the
sensitivity is not as high as claimed.
Righetti G, et al. Mass Spectrom Rev, 2002, 21, 287-302
Disadvantages

2. Though charge-matched, the covalently
modified proteins generated by DIGE have
slightly altered protein migration properties
relative to the bulk of the unlabeled material,
because of the additional mass of the dyes.
Righetti G, et al. Mass Spectrom Rev, 2002, 21, 287-302
Disadvantages






3. One cannot simply run a DIGE gel and cut out the
spots for direct MS analysis.
The real centroid of the spot will not be aligned with
the fluorescent spot.
The vast majority of the spots will be present in too
low an amount to be directly amenable to MS analysis
There is no way to predict where the covalent
fluorescent label will be attached, so that peptide
identification might be problematic.
After the gel has been removed from the
special scanner for fluorescence, the spots will no
longer be visible, and cutting them out will simply be
impossible.
Righetti G, et al. Mass Spectrom Rev, 2002, 21, 287-302
Absolute Quantitation
Barnidge DR, et al. Anal Chem 2003, 75, 445-51
Gerber SA, et al. PNAS, 2003, 100,6940-5
Visible Isotope-Coded Affinity
Tags
Lu Y, et al. Anal Chem, 2004, 76,4104-4111
ITRAQ