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Quantitative proteomics

Quantitative proteomics. Peking Union Medical College Chinese Academy of Medical Sciences Wei Sun sunwei1018@sina.com. Content. 1.Introduction 2. MS-based methods 3. Gel-based methods. Petterson SD, et al. Nat Genetics, 2003, 33, 311-23.

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Quantitative proteomics

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  1. Quantitative proteomics Peking Union Medical College Chinese Academy of Medical Sciences Wei Sun sunwei1018@sina.com

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

  3. Petterson SD, et al. Nat Genetics, 2003, 33, 311-23

  4. Petterson SD, et al. Nat Genetics, 2003, 33, 311-23

  5. 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

  6. 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)

  7. Petterson SD, et al. Nat Genetics, 2003, 33, 311-23

  8. 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

  9. The measurement of the celluar response to external perturbations at the mRNA and protein level are complementary

  10. 6200-997-289 Ideker T, et al. Science,2001,292,929-934

  11. Applications of Quantitative Proteomics • Indentify differenial expressed protein in different states • Detect alternation in protein post-translational modification • Protein complex characterization • Protein-protein interactions

  12. Quantitative proteomics analysis of yeast grown in ethanol versus galactose Gygi et al. Nature Biotech, 1999, 17:994-9

  13. Gygi et al. Nature Biotech, 1999, 17:994-9

  14. Quantitative proteomic analysis of Myc oncoprotein function Shiio Y, EMBO, 2002,21,5088-96

  15. 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

  16. MS-based methods • 1.Separation: 2D-LC/MS/MS (SCX-RP) • 2. Identification: mass spectrometry and database searching algorithm • 3. Label: chemical probes

  17. MS-based methods Yates JR, et al. Nat Biotech, 2001, 19,242-7

  18. MS-based quantitation

  19. 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?

  20. Isotope-coded affinity tags (ICAT) Gygi,S.P.,et al. Nat Biotech, 1999, 17, 994-9

  21. 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

  22. 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

  23. 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.

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

  25. Aebersold R, et al. Nat Biotech, 2002, 19,512-5

  26. Acid-labile isotope codedextractants (ALICE) Wang JH, et al. Anal Chem, 2002,74,4969-79

  27. Wang JH, et al. Anal Chem, 2002,74,4969-79

  28. 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.

  29. 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

  30. 13C-Isotope-coded Affinity Tag

  31. 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.

  32. Chemical probes Aebersold R, et al. Curr Opin Chem Bio, 2004, 8, 66-75

  33. N-terminus Liebler DC, et al. J Proteome Res, 2003, 2, 265-72

  34. James P, et al.Anal Chem, 2000, 72, 4047-57

  35. C-terminus Fenselau C, et al. Anal Chem, 2001, 73, 2836-42

  36. Tryptophan Nishimura O, et al.Rapid Commun Mass Spectrom, 2003, 17, 1642-50

  37. Mass-coded abundance tagging (MCAT) Emili A, et al.Nat Biotech,2002, 20, 163-70

  38. Reilly JP, et al. Rapid Commun Mass Spectrom, 2000, 14, 2147-53

  39. Emili A, et al.Nat Biotech,2002, 20, 163-70

  40. Element-Coded Affinity Tags (ECAT) Whetstone PA, et al.Bioconjugate Chem, 2004,15, 3-6

  41. 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.

  42. Cell Culture Fu EW, et al. Rapid Commun Mass Spectrom, 2002, 16, 1389-97

  43. 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.

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