AGP nLC-MS/MS and nLC-FAIMS-MS/MS

Bottom-up nLC-MS/MS glycoprotein mass spectrometry workflows rely on the generation of a mixture of unmodified and glycosylated peptides via proteolysis. Such methods are challenged by suppression of hydrophilic glycopeptide ions by more abundant, hydrophobic, and readily ionizable unmodified peptides. Commercially available high-field asymmetric waveform ion mobility spectrometry (FAIMS) devices have recently been introduced and present an opportunity for orthogonal separation following chromatic graphic separation and prior to MS/MS analysis, with potential benefits for glycoproteomic workflows. However, knowledge is lacking regarding the optimal FAIMS conditions for glycopeptide analysis. Here, we document optimal FAIMS compensation voltages for the transmission and analysis of human alpha-1-acid glycoprotein (AGP) tryptic N-glycopeptide ions. Further, we evaluate the effect of FAIMS on AGP glycopeptide assignments by comparing the number of assignments at different confidence intervals using a standard nLC-MS/MS method to an identical method with FAIMS.