quantitative proteomics peking union medical college chinese academy of medical sciences wei sun...
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Quantitative proteomicsQuantitative proteomics
Peking Union Medical College
Chinese Academy of Medical Sciences
Wei Sun
ContentContent
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 ProteomicsExpression ProteomicsExpression 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 ProteomicsFunction 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
IntroductionIntroduction1. Quantitation proteomics:
The global analysis of protein expression, a complementary method to study steady-state gene expression and perturbation-induced changes.
Gygi,S.P,et al. Nat Biotech, 1999, 17, 994-9
The measurement of the celluar response The measurement of the celluar response to external perturbations at the mRNA to external perturbations at the mRNA and protein level are complementaryand protein level are complementary
Ideker T, et al. Science,2001,292,929-9346200-997-289
Applications of Quantitative ProteomicsApplications of Quantitative Proteomics
Indentify differenial expressed protein in different states
Detect alternation in protein post-translational modification
Protein complex characterizationProtein-protein interactions
Quantitative proteomics analysis of yeast Quantitative proteomics analysis of yeast grown in ethanol versus galactosegrown 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
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
ApplicationApplication
MS-based methodsMS-based methods
1.Separation: 2D-LC/MS/MS (SCX-RP)
2. Identification: mass spectrometry and database searching algorithm
3. Label: chemical probes
MS-based methodsMS-based methods
Yates JR, et al. Nat Biotech, 2001, 19,242-7
MS-based quantitationMS-based quantitation
Chemical probesChemical 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
AdvantagesAdvantages 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
DisadvantagesDisadvantages 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 post-translational modifications.
Gygi,S.P.,et al. Nat Biotech, 1999, 17, 994-9
Solution(1)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 post-translational modifications.
Solid-phase isotope taggingSolid-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 codedextractants (ALICE)
Wang JH, et al. Anal Chem, 2002,74,4969-79
Wang JH, et al. Anal Chem, 2002,74,4969-79
Solution(2)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 post-translational 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)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 post-translational modifications.
Aebersold R, et al. Curr Opin Chem Bio, 2004, 8, 66-75
Chemical probes
N-terminusN-terminus
Liebler DC, et al. J Proteome Res, 2003, 2, 265-72
James P, et al.Anal Chem, 2000, 72, 4047-57
C-terminusC-terminus
Fenselau C, et al. Anal Chem, 2001, 73, 2836-42
TryptophanTryptophan
Nishimura O, et al.Rapid Commun Mass Spectrom, 2003, 17, 1642-50
Mass-coded abundance Mass-coded abundance tagging (MCAT)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)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 post-translational modifications.
Cell CultureCell 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. MCP2002, 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 database-searching programs and prevent protein identification prior to quantification.
Gygi SP, et al. Curr Opin Biotech. 2000,11,396-401
SolutionSolution 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 post-translational 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, 2071–2077
Advantages1. 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
Disadvantages1. 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
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.
Disadvantages
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
