Abstract
We have applied liquid chromatography high-field asymmetric waveform ion mobility spectrometry tandem mass spectrometry (LC-FAIMS-MS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) to the investigation of site-specific phosphorylation in fibroblast growth factor (FGF) signaling. We have combined a SILAC approach with chemical inhibition by SU5402 (an FGF receptor tyrosine kinase inhibitor) and dasatinib (a Src family kinase inhibitor). The results show that incorporation of FAIMS within the workflow results in (a) an increase in the relative proportion of phosphothreonine and phosphotyrosine sites identified, (b) an increase in phosphopeptide identifications from precursors with charge states ≥ +3 (with an associated increase in peptide length), and (c) an increase in the identification of multiply phosphorylated peptides. Approximately 20% of the phosphorylation sites identified via the FAIMS workflow had not been reported previously, and over 80% of those were from multiply phosphorylated peptides. Moreover, FAIMS provided access to a distinct set of phosphorylation sites regulated in response to SU5402 and dasatinib. The enhanced identification of multiply phosphorylated peptides was particularly striking in the case of sites regulated by SU5402. In addition to providing a compelling example of the complementarity of FAIMS in phosphoproteomics, the results provide a valuable resource of phosphorylation sites for further investigation of FGF signaling and trafficking.
Highlights
Fibroblast growth factor (FGF) ligands and receptors play significant roles in cell division, regulation of cell growth and maturation, angiogenesis, wound healing, and tumor growth.[1]Accumulating evidence suggests that deregulation of FGF signaling is associated with many human diseases, including cancer.[2]
It has previously been shown that LC−MS/ MS and LC−field asymmetric waveform ion mobility spectrometry (FAIMS)−MS/MS are complementary in their coverage of the proteome,[18] and it is this complementarity that we wished to exploit in order to extend the phosphoproteomic coverage of components of the FGF signaling pathway
Our LC−MS/MS and LC−FAIMS−MS/MS analyses combined SILAC labeling with strong cation exchange (SCX) prefractionation and phosphoenrichment
Summary
Accumulating evidence suggests that deregulation of FGF signaling is associated with many human diseases, including cancer.[2] Through phosphorylation and dephosphorylation processes, FGF signaling is propagated through receptor proteins, scaffolding proteins, and signal mediators, leading to a number of downstream pathways.[3] Of particular interest, the nonreceptor tyrosine kinase Src has been demonstrated to play a critical role in downstream FGFR trafficking.[4]. Considering their broad participation in signal transduction, it is not surprising that phosphorylation events hold the key to understanding signaling events downstream of FGFR. Functional interpretation of phosphorylation requires detailed analysis of specific residues or combinations of residues
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