Protein N-glycosylation is essential for mammalian cell survival and is well-known to be involved in many biological processes. Aberrant glycosylation is directly related to human disease including cancer and infectious diseases. Global analysis of protein N-glycosylation will allow a better understanding of protein functions and cellular activities. Mass spectrometry (MS)-based proteomics provides a unique opportunity to site-specifically characterize protein glycosylation on a large scale. Due to the complexity of biological samples, effective enrichment methods are critical prior to MS analysis. Here, we compared two lectin-independent methods to enrich glycopeptides for the global analysis of protein N-glycosylation by MS. The first boronic acid-based enrichment (BA) method benefits from the universal and reversible interactions between boronic acid and sugars; the other method utilizes metabolic labeling and click chemistry (MC) to incorporate a chemical handle into glycoproteins for future affinity enrichment. We comprehensively compared the performance of the two methods in the identification and quantification of glycoproteins in statin-treated liver cells. Based on the current results, the BA method is more universal in enriching glycopeptides, while with the MC method, cell surface glycoproteins were highly enriched, and the quantification results appear to be more dynamic because only the newly-synthesized glycoproteins were analyzed. In addition, we normalized the glycosylation site ratios by the corresponding parent protein ratios to reflect the real modification changes. In combination with MS-based proteomics, effective enrichment methods will vertically advance protein glycosylation research.

中文翻译：

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使用两种不依赖凝集素的化学富集方法对他汀处理的肝细胞中的 N-糖蛋白组进行质谱分析

蛋白质 N-糖基化对于哺乳动物细胞的存活至关重要，并且众所周知参与许多生物过程。异常糖基化与包括癌症和传染病在内的人类疾病直接相关。蛋白质 N-糖基化的全局分析将有助于更好地了解蛋白质功能和细胞活动。基于质谱 (MS) 的蛋白质组学为大规模定点表征蛋白质糖基化提供了独特的机会。由于生物样品的复杂性，有效的富集方法在 MS 分析之前至关重要。在这里，我们比较了两种不依赖凝集素的方法来富集糖肽，以通过 MS 对蛋白质 N-糖基化进行全局分析。第一种基于硼酸的富集 (BA) 方法受益于硼酸和糖之间普遍且可逆的相互作用；另一种方法利用代谢标记和点击化学 (MC) 将化学处理结合到糖蛋白中，用于未来的亲和力富集。我们全面比较了两种方法在他汀类药物处理的肝细胞中糖蛋白的鉴定和定量方面的性能。根据目前的结果，BA 方法在富集糖肽方面更为普遍，而 MC 方法则高度富集细胞表面糖蛋白，并且由于只分析了新合成的糖蛋白，因此定量结果似乎更具动态性。此外，我们通过相应的亲本蛋白比例对糖基化位点比例进行了标准化，以反映真实的修饰变化。结合基于 MS 的蛋白质组学，有效的富集方法将垂直推进蛋白质糖基化研究。