Glycosylation is a ubiquitous post-translational modification (PTM) that strongly affects the protein folding and function. Glycosylation patterns are impacted by many diseases, making promising biomarkers. Glycans are also the most complex PTMs, exhibiting isomers (linkage, anomers, and those with isomeric moieties). Permuted with localization variants that occur for all PTMs, these produce numerous isomeric glycoforms. Characterizing them by mass spectrometry and ion mobility spectrometry (IMS) has been a challenge. High-definition differential IMS (FAIMS) had robustly disentangled isomeric peptides involving other PTMs but was not evaluated for glycopeptides that featured multilevel isomerism. Here, we apply it to representative mucin glycopeptides with O-linked glycans: three GalNAc localization variants, a pair with α/β GalNAc anomers, and another with GalNAc/GlcNAc isomers. The first two classes were separated baseline with the resolution exceeding previous benchmarks by 10-fold, and the last pair was partly resolved. The recently demonstrated straightforward coupling to ultrahigh-resolution MS and electron-transfer dissociation makes high-definition FAIMS an attractive tool for glycoproteomics.

中文翻译：

---

异构体糖形的高分辨率离子淌度分离，其肽和糖基水平存在差异。

糖基化是一种普遍存在的翻译后修饰（PTM），会强烈影响蛋白质的折叠和功能。糖基化模式受到许多疾病的影响，成为有前途的生物标志物。聚糖也是最复杂的PTM，表现出异构体（键，异构体和具有异构部分的异构体）。与所有PTM都发生的定位变体排列在一起，它们会产生许多异构糖型。通过质谱和离子迁移谱（IMS）对其进行表征一直是一个挑战。高清差分IMS（FAIMS）具有涉及其他PTM的牢固解开的异构肽，但未评估具有多级异构性的糖肽。在这里，我们将其应用于具有O型连接聚糖的代表性粘蛋白糖肽：三个GalNAc定位变体，一对与α/βGalNAc异构体，另一个是GalNAc / GlcNAc异构体。前两个类别的基准线分离，分辨率比以前的基准高出十倍，最后一对被部分解决。最近证明的与超高分辨率质谱和电子转移解离的直接偶联使高清FAIMS成为糖蛋白组学的诱人工具。