A fully automated online enrichment and separation system for intact glycopeptides, named AutoGP, was developed in this study by integrating three different columns in a nano-LC system. Specifically, the peptide mixture from the enzymatic digestion of a complex biological sample was first loaded on a hydrophilic interaction chromatography (HILIC) column. The nonglycopeptides in the sample were washed off the column, and the glycopeptides retained by the HILIC column were eluted to a C18 trap column to achieve an automated glycopeptide enrichment. The enriched glycopeptides were further eluted to a C18 column for separation, and the separated glycopeptides were eventually analyzed by using an orbitrap mass spectrometer (MS). The optimal operating conditions for AutoGP were systemically studied, and the performance of the fully optimized AutoGP was compared with a conventional manual system used for glycopeptide analysis. The experimental evaluation shows that the total number of glycopeptides identified is at least 1.5-fold higher, and the median coefficient of variation for the analyses is at least 50% lower by using AutoGP, as compared to the results acquired by using the manual system. In addition, AutoGP can perform effective analysis even with a 1-μg sample amount, while a 10-μg sample at least will be needed by the manual system, implying an order of magnitude better sensitivity of AutoGP. All the experimental results have consistently proven that AutoGP can be used for much better characterization of intact glycopeptides.

中文翻译：

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全自动在线富集和分离系统，可对完整糖肽进行高度重复和深入的分析

本研究通过将三种不同的色谱柱集成到纳米液相色谱系统中，开发了一种完整糖肽的全自动在线富集和分离系统，称为 AutoGP。具体来说，首先将复杂生物样品酶消化得到的肽混合物加载到亲水相互作用色谱 (HILIC) 柱上。将样品中的非糖肽从柱上洗掉，将HILIC柱保留的糖肽洗脱到C18捕获柱上，实现糖肽的自动化富集。富集的糖肽进一步洗脱到C18柱上进行分离，最终使用轨道阱质谱仪（MS）对分离的糖肽进行分析。系统研究了 AutoGP 的最佳操作条件，并将完全优化的 AutoGP 的性能与用于糖肽分析的传统手动系统进行了比较。实验评估表明，与使用手动系统获得的结果相比，使用 AutoGP 识别的糖肽总数至少高出 1.5 倍，分析的中值变异系数至少降低 50%。此外，AutoGP即使在1μg的样品量下也能进行有效分析，而手动系统至少需要10μg的样品，这意味着AutoGP的灵敏度提高了一个数量级。所有实验结果一致证明 AutoGP 可用于更好地表征完整糖肽。