Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of healthy and disease-driven biological functions. Previously, we developed a mucin-selective enrichment strategy by employing a catalytically inactive mucinase (StcE) conjugated to a solid support. While this method was effective, it suffered from low throughput and high sample requirements. Further, the elution step required boiling in SDS, thus necessitating an in-gel digest with trypsin. Here, we introduce innovative elution conditions amenable to mucinase digestion and downstream analysis using mass spectrometry. This increased throughput and lowered sample input while maintaining mucin selectivity and enhancing the glycopeptide signal. We then benchmarked this technique against different O-glycan binding moieties for their ability to enrich mucins from various cell lines and human serum. Overall, the new method outperformed our previous procedure and all of the other enrichment techniques tested. This allowed for the effective isolation of more mucin-domain glycoproteins, resulting in a high number of O-glycopeptides, thus enhancing our ability to analyze the mucinome.

中文翻译：

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质谱兼容的洗脱技术实现了改进的粘蛋白选择性富集策略来探测粘蛋白组

粘蛋白结构域糖蛋白高度 O-糖基化，在许多健康和疾病驱动的生物功能中发挥着关键作用。此前，我们通过采用与固体支持物缀合的催化失活粘蛋白酶（StcE）开发了粘蛋白选择性富集策略。虽然该方法有效，但其通量低且样品要求高。此外，洗脱步骤需要在 SDS 中煮沸，因此需要用胰蛋白酶进行凝胶内消化。在这里，我们介绍了适合粘蛋白酶消化和使用质谱进行下游分析的创新洗脱条件。这增加了通量并降低了样品输入，同时保持了粘蛋白选择性并增强了糖肽信号。然后，我们针对不同的 O-聚糖结合部分对这项技术进行了基准测试，以确定它们从各种细胞系和人血清中富集粘蛋白的能力。总体而言，新方法优于我们之前的程序和测试的所有其他富集技术。这使得能够有效分离更多的粘蛋白结构域糖蛋白，从而产生大量的O-糖肽，从而增强我们分析粘蛋白组的能力。