Protein kinase C’s are distributed in different compartment and found at the nexus of the many signalling pathways. PKCs regulate many vital pathways required for cell survival, proliferation and metabolism. Any aberrant alteration in the PKC structure/sequence/chemical modification leads to manifestation of many diseases ranging from inflammation to cancer. Recently, the compartment specific functions of these PKC isoforms are attributed to the post-translational modifications (PTM) at specific sites. PTM plays an important role in regulating their activation and translocation in different compartments of the cell. Most of the study of PKC PTM are done in non-endogenous conditions so necessarily not reflect the natural environment with in the cell. Here, we are trying to isolate endogenous PKC using membrane preparation and cell surface labelling methods.

Mass spectrometry has evolved dramatically, and is now considered a key method for site mapping, quantification of chemical modifications (PTM) and even for detecting protein–protein interactions. The right combination of MS method with maximum enrichment is crucial for mapping of proteins to study their PTMs in specific cell compartment.

Cell surface labelling helps to separate the cell surface protein by labelling the surface protein with biotin non-specifically followed by affinity purification using a neutravidin column. Membrane preparation will enrich the membrane proteins including membrane associated protein like protein kinase C’s by using differential ultracentrifugation. By combining these two methods, selective enrichment of low abundant proteins like PKC’s can be done. Selectively enriched membrane or membrane associated proteins can then be characterised for its PTM study by new MS method like SWATH.