Evaluation and Optimization of High-Field Asymmetric Waveform Ion Mobility Spectrometry for Multiplexed Quantitative Site-specific N-glycoproteomics

The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N glycopeptides derived from the purified IgM protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by N glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size and precursor m/z. Importantly, FAIMS also improved multiplexed N glycopeptide quantification, both with the standard MS2 acquisition method and with our recently developed Glyco-SPS-MS3 method. The combination of FAIMS and Glyco-SPS-MS3 provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass-spectrometry-based qualitative and quantitative N glycoproteomics.