1. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS Gygi et al (2003) PNAS 100(12), 6940-6945. presented by Jessica Lee for MEDG 505

2. Outline Introduction Methods & Results Conclusions Critiques Discussion Topics

3. Central dogma of biology

4. Protein modification ModificationTarget site(s)Cellular process PhosphorylationS, T, YSignalling, activation MethylationR, K, H, GProtein repair, chemotaxis AcetylationK, N-terminusGene expression UbiquitylationKDegradation SulfationYProtein-protein interaction PalmitoylationCysMembrane association

5. Previous methods Relative quantification  2-dimensional gel electrophoresis  tandem MS Absolute quantification  use of synthetic peptides with the stable isotopes incorporated.

6. Previous methods Two-dimensional gel electrophoresis  quantification by protein staining  comparative analysis  protein identified by MS  limited to abundant proteins

7. Previous methods Tandem MS  protein and peptide separation coupled with amino acid sequence analysis  quantification by stable isotopes labelling  mass accuracy resolution reliability of the mass calibration relationship

8. Previous methods Absolute protein quantification with synthetic peptide  stable isotopes incorporation  liquid chromatography (LC) – MS  selected reaction monitoring (SRM) extreme sensitivity target -> precursor ion -> production ion

9. Methods

10. Part 1: AQUA internal standard peptide chose peptide based on  amino acid sequence  protease solid phase peptide synthesis incorporate stable isotopes evaluate peptide by LC- MS/MS AQUA = absolute quantification

11. Part 2: method development & implemmentation separate whole cell lysate by SDS/PAGE gel electrophoresis in gel digestion with the AQUA peptides LC-SRM determine precise expression level from LC- MS/MS

12. Validations Purified protein Target protein in whole cell lysate Trypsinization Low-abundance proteins in whole cell lysate Posttranslational modified protein - phosphorylation

13. Conclusions Sensitivity and specificity Sample requirement Cover the short comings of previous methods Assist the study of systems biology in diverse ways Insights into cellular events and pathways

14. Critiques Lack of information  introduction  discussion limitation Short comings of previous methods Complement to lack of protein abundance measurement

15. Discussion topics Limitations of this methods  known proteins  Y-type fragment ions for SRM Improvements  high throughput For unknown proteins