Journal
ANALYTICAL CHEMISTRY
Volume 93, Issue 25, Pages 8846-8855Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c00802
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Funding
- Manfred Eigen Fellowship from the Max Planck Institute for Biophysical Chemistry
- Collaborative Research Center of the Deutsche Forschungsgemeinschaft [SFB1286]
- LOEWE Center Frankfurt Cancer Institute (FCI) - Hessen State Ministry for Higher Education, Research and the Arts [III L 5-519/03/03.001-(0015)]
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The heterogeneity and complexity of glycosylation have posed challenges for site-specific glycoproteomics analysis. The use of FAIMS has been shown to enhance the scope of bottom-up proteomics but its benefits for quantitative N-glycoproteomics have not been extensively studied. In this work, optimized FAIMS settings improved the identification and quantification of site-specific N-glycopeptides, demonstrating the advantages of FAIMS for both qualitative and quantitative N-glycoproteomics analysis.
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 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 immunoglobulin M (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 methods provided the highest quantitative accuracy and precision. Our results demonstrate the advantages of FAIMS for improved mass spectrometry-based qualitative and quantitative N-glycoproteomics.
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