Question

9. You have a highly purified protein. You want to assess its post translational modification pattern. What type of mass-spectrometry would you use and what ionisation source do you think would be most appropriate? Justify your answer (500 words)

Answer 1

Solution: The post translational modifications include the covalent, enzyme based modifications of proteins occuring in various sub-cellular organelles like endoplasmic reticulum, golgi etc. The modifications include N and O glycosylations, proteolytic cleavages, sulphur linkage formation by protein disulphide isomerases, ubiqutination etc. These modifications are necessary for the stability and functioning of the proteins.

Post translational modifications of proteins can be detected through various techniques like Western blotting, Mass spectrometry etc. In mass spectoremetry mediated detection assay, initially we need to immuno precipitate the proteins. The. Immuno precipitation allows us to analyse low abundance post translational modifications on the target protein. This is done through affinity based purification processes In the majority of mass spectrometric studies, on post translational modifications it is desirable to use immuno precipitation. The mass spectrometry measures the mass to charge ratio of ions. We obtain the resukts in the form of a mass spectrum. Here we can identify a huge range of proteins undergone post translational modifications using bottom up peptide based mass spectrometric analysis or top down mass spectrometric analysis. This is a broad spectrum analysis method in which we can identify the modifications on huge range of proteins. This also helps identify specific site targeted modifications. Also there is no need of primary antibody for detection.