Quantitative proteomicsQuantitative proteomics

Peking Union Medical College

Chinese Academy of Medical Sciences

Wei Sun

sunwei1018@sina.com

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

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