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

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell, Cell Culture

DISEASE(S): Lymphoma

SUBMITTER: Pan Fang  

LAB HEAD: Kuan-Ting Pan

PROVIDER: PXD023951 | Pride | 2021-05-05

REPOSITORIES: Pride