Developing a LC-MRM method to screen for the abundance and phosphorlation state of specific Src kinases associated with the IL-6 signal transducer, gp130

Objective: To develop a Liquid Chromatography-Multiple Reaction Monitoring (LC-MRM) based method to simultaneously quantify Src Family Kinase (SFK) expression and phosphorylation state, we derived a list of tryptic digested peptides in silico with Skyline software for each SFK member. Peptides will be selected for synthesis and future quantification of SFK expression and phosphorylation based on how each peptide demonstrates consistent detection with strong relative intensity by LC-MRM Results: Unique peptides were chosen for each SFK to be used to differentiate individual family members. Two common peptides were also selected to bifurcate the SFK family into two groups. In addition, peptides containing the phosphorylated tyrosine residue were selected to monitor SFK activation state. Based on these results, peptides were synthesized and will be used as internal standards to quantitatively measure expression and phosphorylation of SFK. Conclusion: This work will establish a new LC-MRM peptide based method to simultaneously quantify the abundance and phosphorylation of the SFK kinome in very small sample sizes that are not amenable to analysis by other methods. As such, this technique will have a broad range of experimental applications and will be particularly useful in future studies that validate results from cell line models with patient specimens