Improvement of Phosphoproteome Analyses Using FAIMS and Decision Tree Fragmentation. Application to the Insulin Signaling Pathway in Drosophila melanogaster S2 Cells

This report examines the analytical benefits of high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to liquid chromatography mass spectrometry (LC–MS) for phosphoproteomics analyses. The ability of FAIMS to separate multiply charged peptide ions from chemical interferences confers a unique advantage in phosphoproteomics by enhancing the detection of low abundance phosphopeptides. LC–FAIMS–MS experiments performed on TiO2-enriched tryptic digests from Drosophila melanogaster provided a 50% increase in phosphopeptide identification compared to conventional LC–MS analysis. Also, FAIMS can be used to select different population of multiply charged phosphopeptide ions prior to their activation with either collision activated dissociation (CAD) or electron transfer dissociation (ETD). Importantly, FAIMS enabled the resolution of coeluting phosphoisomers of different abundances to facilitate their unambiguous identification using conventional database search engines. The benefits of FAIMS in large-scale phosphoproteomics of D. melanogaster are further investigated using label-free quantitation to identify differentially regulated phosphoproteins in response to insulin stimulation.