Monday, April 16, 2012

Identification of Targets of c-Src Tyrosine Kinase by Chemical Complement and Phosphoproteomics

This new paper in press at MCP by Martinez-Ferrando, et al., is a really good example of using cutting edge phosphoproteomics to open up our understanding of an incredibly interesting protein.  Although c-Src has been the focus of thousands of different papers, its amazing how much we still don't know about a protein that was discovered in the 1970s (and resulted in a Nobel prize for 2 top scientists in the 1980s).

In this study, a system was established to force the c-Src tyrosine kinase function to be active in a SILAC labeling system.  An interesting aspect of this study is that they only went for peptides phosphorylated on tyrosine residues, by use of the filter aided capture and elution (FACE) using the 4G10 antibody.  They didn't follow up with a metal or ion based phosphopeptide enrichment.

The proteomic analysis was top-notch, as you'd expect from the Pandey lab, using an Orbitrap Velos in 'high high' mode, where they used the Orbitrap exclusively, both collecting the MS1 and MS/MS fragmentation information (via HCD).  Both Proteome Discoverer 1.3 and Maxquant were employed because they often provide complementary information. My only criticism of this excellent paper is that peptide desalting was performed with C-18, which causes a loss of phosphopeptides, compared to other desalting methods such as graphite spin columns.

The results of this study are over 200 proteins that were shown to be downstream phosphorylation events that were downstream of c-Src.  The majority of these phosphorylations were previously unknown to be linked to c-Src and help explain some of the effects of this important oncogene.  They do a good job of 'validating' several of these events by western blotting and immunocytochemistry (ICC).

Summary:  A really good paper that shows how phosphoproteomics can really open our eyes, even when it comes to extremely well studied and characterized systems.  A good read for anyone in oncology research.