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FAIMS and Phosphoproteomics of Fibroblast Growth Factor Signaling: Enhanced Identification of Multiply Phosphorylated Peptides
journal contribution
posted on 2015-12-09, 06:49 authored by Hongyan Zhao, Debbie L. Cunningham, Andrew J. Creese, John K. Heath, Helen J. CooperWe
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.
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Fibroblast Growth Factor SignalingSrc family kinase inhibitorphosphorylation sitesSILACchromatography tandem mass spectrometryinvestigationidentificationworkflowwaveform ion mobility spectrometry tandem mass spectrometryFAIMSdasatinibfibroblast growth factorFGF receptor tyrosine kinase inhibitorSU 5402phosphorylated peptidesLC
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