Rangel-Angarita Valentina, Mahoney Keira E, Kwon Catherine, Sarker Raibat, Lucas Taryn M, Malaker Stacy A
Department of Chemistry, Yale University, 275 Prospect Street, New Haven, Connecticut 06511, United States.
JACS Au. 2023 Sep 12;3(9):2498-2509. doi: 10.1021/jacsau.3c00264. eCollection 2023 Sep 25.
High-field asymmetric waveform ion mobility spectrometry (FAIMS) separates glycopeptides in the gas phase prior to mass spectrometry (MS) analysis, thus offering the potential to analyze glycopeptides without prior enrichment. Several studies have demonstrated the ability of FAIMS to enhance glycopeptide detection but have primarily focused on N-glycosylation. Here, we evaluated FAIMS for O-glycoprotein and mucin-domain glycoprotein analysis using samples of varying complexity. We demonstrated that FAIMS was useful in increasingly complex samples as it allowed for the identification of more glycosylated species. However, during our analyses, we observed a phenomenon called "in FAIMS fragmentation" (IFF) akin to in source fragmentation but occurring during FAIMS separation. FAIMS experiments showed a 2- to 5-fold increase in spectral matches from IFF compared with control experiments. These results were also replicated in previously published data, indicating that this is likely a systemic occurrence when using FAIMS. Our study highlights that although there are potential benefits to using FAIMS separation, caution must be exercised in data analysis because of prevalent IFF, which may limit its applicability in the broader field of O-glycoproteomics.
高场不对称波形离子迁移谱（FAIMS）在质谱（MS）分析之前于气相中分离糖肽，从而提供了无需预先富集即可分析糖肽的潜力。多项研究已证明FAIMS增强糖肽检测的能力，但主要集中在N-糖基化方面。在此，我们使用不同复杂程度的样品评估了FAIMS用于O-糖蛋白和粘蛋白结构域糖蛋白分析的情况。我们证明，FAIMS在日益复杂的样品中很有用，因为它能够鉴定出更多的糖基化种类。然而，在我们的分析过程中，我们观察到一种类似于源内裂解但发生在FAIMS分离过程中的现象，称为“FAIMS内裂解”（IFF）。FAIMS实验表明，与对照实验相比，IFF产生的光谱匹配增加了2至5倍。这些结果在先前发表的数据中也得到了重复，表明使用FAIMS时这可能是一种系统性现象。我们的研究强调，尽管使用FAIMS分离有潜在益处，但由于普遍存在的IFF，在数据分析时必须谨慎，这可能会限制其在更广泛的O-糖蛋白质组学领域中的适用性。
