Zhao Hongyan, Cunningham Debbie L, Creese Andrew J, Heath John K, Cooper Helen J
School of Biosciences, University of Birmingham , Edgbaston, Birmingham B15 2TT, United Kingdom.
J Proteome Res. 2015 Dec 4;14(12):5077-87. doi: 10.1021/acs.jproteome.5b00713. Epub 2015 Nov 5.
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.
我们已将液相色谱-高场非对称波形离子淌度谱串联质谱（LC-FAIMS-MS/MS）和液相色谱串联质谱（LC-MS/MS）应用于成纤维细胞生长因子（FGF）信号通路中位点特异性磷酸化的研究。我们将稳定同位素标记氨基酸细胞培养法（SILAC）与SU5402（一种FGF受体酪氨酸激酶抑制剂）和达沙替尼（一种Src家族激酶抑制剂）的化学抑制相结合。结果表明，在工作流程中加入FAIMS会导致：（a）所鉴定的磷酸苏氨酸和磷酸酪氨酸位点的相对比例增加；（b）来自电荷态≥ +3的前体的磷酸肽鉴定增加（同时肽长度也相应增加）；（c）多重磷酸化肽的鉴定增加。通过FAIMS工作流程鉴定出的磷酸化位点中，约20%此前未被报道，其中超过80%来自多重磷酸化肽。此外，FAIMS还能鉴定出一组受SU5402和达沙替尼调控的独特磷酸化位点。在受SU5402调控的位点中，多重磷酸化肽的鉴定增强尤为显著。这些结果不仅为FAIMS在磷酸蛋白质组学中的互补性提供了一个有说服力的例子，还为进一步研究FGF信号传导和运输提供了一份宝贵的磷酸化位点资源。
