Fang Pan, Ji Yanlong, Silbern Ivan, Viner Rosa, Oellerich Thomas, Pan Kuan-Ting, Urlaub Henning
Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Hematology/Oncology, Department of Medicine II, Johann Wolfgang Goethe University, 60590 Frankfurt am Main, Germany.
Anal Chem. 2021 Jun 16. doi: 10.1021/acs.analchem.1c00802.
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 -glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for -glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific -glycopeptides derived from the purified immunoglobulin M (IgM) protein or human lymphoma cells. We explored in detail the changes in FAIMS mobility caused by -glycopeptides with different characteristics, including TMT labeling, charge state, glycan type, peptide sequence, glycan size, and precursor /. Importantly, FAIMS also improved multiplexed -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 -glycoproteomics.
糖基化的异质性和复杂性阻碍了位点特异性糖蛋白质组学分析的深度。高场非对称波形离子迁移谱（FAIMS）已被证明可拓宽自下而上蛋白质组学的范围。FAIMS在定量糖蛋白质组学方面的优势尚未得到研究。在这项工作中，我们针对有或没有串联质量标签（TMT）标记的糖肽鉴定优化了FAIMS设置。优化后的FAIMS方法显著增加了从纯化的免疫球蛋白M（IgM）蛋白或人淋巴瘤细胞中鉴定出的位点特异性糖肽。我们详细探究了具有不同特征的糖肽（包括TMT标记、电荷状态、聚糖类型、肽序列、聚糖大小和前体/）引起的FAIMS迁移率变化。重要的是，无论是使用标准的MS2采集方法还是我们最近开发的Glyco-SPS-MS3方法，FAIMS都改善了多重糖肽定量。FAIMS和Glyco-SPS-MS3方法的结合提供了最高的定量准确性和精密度。我们的结果证明了FAIMS在改进基于质谱的定性和定量糖蛋白质组学方面的优势。
