Quantitative proteomics Peking Union Medical College Chinese Academy of Medical Sciences Wei Sun

Content 1.Introduction 2. MS-based methods 3. Gel-based methods

Petterson SD, et al. Nat Genetics, 2003, 33,

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,

Introduction 1. 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 to external perturbations at the mRNA and protein level are complementary

Ideker T, et al. Science,2001,292,

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

Quantitative proteomic analysis of Myc oncoprotein function Shiio Y, EMBO, 2002,21,

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 Application

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 post- translational 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 post- translational modifications.

Solid-phase isotope tagging Aebersold R, et al. Nat Biotech, 2002, 19,512-5

Acid-labile isotope coded extractants (ALICE) Wang JH, et al. Anal Chem, 2002,74,

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, Regnier, FE, et al. J Proteome Res, 2002, 1,

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 post- translational modifications.

Aebersold R, et al. Curr Opin Chem Bio, 2004, 8, Chemical probes

N-terminus Liebler DC, et al. J Proteome Res, 2003, 2,

James P, et al.Anal Chem, 2000, 72,

C-terminus Fenselau C, et al. Anal Chem, 2001, 73,

Tryptophan Nishimura O, et al.Rapid Commun Mass Spectrom, 2003, 17,

Mass-coded abundance tagging (MCAT) Emili A, et al.Nat Biotech,2002, 20,

Reilly JP, et al. Rapid Commun Mass Spectrom, 2000, 14,

Emili A, et al.Nat Biotech,2002, 20,

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 post- translational modifications.

Cell Culture Fu EW, et al. Rapid Commun Mass Spectrom, 2002, 16,

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Mann M, et al. MCP 2002, 1, 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,

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 post- translational modifications.

Yates JR, et al. Anal Chem, 2002, 74,

Julka S, et al. J Proteome Res, 2003, 3,

Gel-based methods Aebersold R, et al. MCP, 2002, 1,19-29

Hamdan M, et al. Rapid Commun Mass Spectrom, 2002, 16,

Sechi S, et al. Rapid Commun Mass Spectrom, 2002, 16,

Chemically-coded affinity tag (CCAT) Niehaus K, et al. 2003, J Biotech, 106,

Differential In-gel Electrophoresis (DIGE) Unlu, et al. Electrophoresis 18, 2071–2077

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,

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,

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,

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,

Absolute Quantitation Barnidge DR, et al. Anal Chem 2003, 75,

Gerber SA, et al. PNAS, 2003, 100,6940-5

Visible Isotope-Coded Affinity Tags Lu Y, et al. Anal Chem, 2004, 76,

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