Enhanced Sensitivity and Coverage in Large-Scale Phosphoproteomics Studies using High Field Asymmetric Waveform Ion Mobility Spectrometry and Decision Tree Fragmentation

摘要

FAIMS provides an increase in phosphoproteome coverage in 2D-nanoLC-MS/MS experiments. 2D-nanoLC-FAIMS-MS provided comparable CV values for intensity measurements compared to 2D-nanoLC-MS. Increase of the S/N for low-abundance phosphopeptide enabling confident identification. FAIMS enables the separation of phosphoisomers thereby reducing the occurrence of mixed MS/MS spectra. 50percent and 35percent increase in phosphopeptide and phosphoprotein identification, respectively. Advantages of 2D-nanoLC-FAIMS-MS to highlight differences in S2 cells phosphoproteome after insulin treatment. Identification of positive controls (Phosphoinositide 3-kinase and SAPK-interacting protein 1 significantly up-phosphorylated after insulin treatment and eIF2b significantly down-phosphorylated). Identification of new insulin-dependant phosphorylation events involving cytoskelton (kinesin, Cenp-C) as well as translation and transcription ( eiF3, Spt6, Squid, Rps20, Ilf2...).