Evaluation and Optimization of High-Field Asymmetric Waveform Ion-Mobility Spectrometry for Multiplexed Quantitative Site-Specific <i>N</i> -Glycoproteomics

Abstract

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