Glycosylation is a key modulator of the functional state of proteins. Recent developments in large-scale analysis of intact glycopeptides have enabled the identification of numerous glycan structures that are relevant in pathophysiological processes. However, one motif found in N-glycans, poly-N-acetyllactosamine (polyLacNAc), still poses a substantial challenge to mass spectrometry-based glycoproteomic analysis due to its relatively low abundance and large size. In this work, we developed approaches for the systematic mapping of polyLacNAc-elongated N-glycans in melanoma cells. We first evaluated five anion exchange-based matrices for enriching intact glycopeptides and selected two materials that provided better overall enrichment efficiency. We then tested the robustness of the methodology by quantifying polyLacNAc-containing glycopeptides as well as changes in protein fucosylation and sialylation. Finally, we applied the optimal enrichment methods to discover glycopeptides containing polyLacNAc motifs in melanoma cells and found that integrins and tetraspanins are substantially modified with these structures. This study demonstrates the feasibility of glycoproteomic approaches for identification of glycoproteins with polyLacNAc motifs.

中文翻译：

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通过阴离子交换介导的方法富集的糖肽的质谱分析揭示了黑色素瘤细胞中整合素和四跨膜蛋白中的 PolyLacNAc 延伸的 N-聚糖

糖基化是蛋白质功能状态的关键调节剂。完整糖肽大规模分析的最新进展使得能够鉴定与病理生理过程相关的众多聚糖结构。然而，在N-聚糖中发现的一个基序，聚-N-乙酰基乳糖胺 (polyLacNAc)，由于其相对较低的丰度和较大的尺寸，仍然对基于质谱的糖蛋白质组分析提出了重大挑战。在这项工作中，我们开发了对黑色素瘤细胞中PolyLacNAc 延长的N -聚糖进行系统定位的方法。我们首先评估了五种用于富集完整糖肽的基于阴离子交换的基质，并选择了两种提供更好总体富集效率的材料。然后，我们通过量化含 polyLacNAc 的糖肽以及蛋白质岩藻糖基化和唾液酸化的变化来测试该方法的稳健性。最后，我们应用最佳富集方法在黑色素瘤细胞中发现含有polyLacNAc基序的糖肽，并发现整合素和四跨膜蛋白被这些结构显着修饰。这项研究证明了糖蛋白组学方法鉴定具有 PolyLacNAc 基序的糖蛋白的可行性。