Answers for group discussion paper 3

1. Benefits: (i) Radio isotopes of Carbon and hydrogen are weak so antibody based western blotting can be used for PTM detection. (ii) Isolation of PTM peptides by immuno- affinity based method is easier than the Trypsin based method. (iii) pan-specific antibodies, antibodies specific to PTM motifs in peptides have been used to identify kinase substrates and quantify their PTM changes, the same approach should be applicable for identifying novel substrates where a consensus PTM motif exists for a PTM regulatory enzyme of interest. Drawbacks: (i) High quality antibodies are not available for PTM of interest due to the small size of the motifs. (ii) Quality control of antibody based affinity enrichment is difficult. 2. (i) Subcellular location of protein is differs in each organelles and protein complexes. (ii) The protein stoichiometry differs based on the molecular and cellular context. 3. Proteolytic digestion causes the artificial PTMs. 4. They can be distinguished by using the quantitative MS methods. 5. Labeling of peptides with stable isotopes can be achieved either in vitro or in vivo. Isotopic labeling methods offer the advantage of higher accuracy and 6. It facilitates easy and efficient fragmentation of large peptides and small protein domain and it is becomes feasible to study multi-site and cooperative PTMs within small protein domains. 7. It will reduce the protein sample complexity and keep the proteins biologically active. 8. Separate the mono phosphorylated peptides from the multi phosphorylated so it reduces the sample complexity and also further improve our abilities to pursue comprehensive phosphoproteome analysis in the future.