Protein glycosylation is the attachment of a carbohydrate moiety to a protein backbone affecting both structure and function of the protein. Abnormal glycosylation is associated with various diseases, and some of the changes in glycosylation are detectable even before symptom development. As such, glycans have emerged as compelling new biomarker candidates. A wide range of analytical methods exist for small-scale glycan analyses. However, there is a growing need for highly robust and reproducible high-throughput techniques that allow for large-scale glycoprofiling. Here, we describe the evaluation of robustness and repeatability of immunoglobulin G (IgG) N-glycan analysis using the GlycoWorks RapiFluor-MS N-Glycan Kit followed by hydrophilic interaction ultra-high-performance liquid chromatography (HILIC-UHPLC) from 335 technical replicates of human plasma randomly distributed across 67 96-well plates. The data was collected over a 5-month period using multiple UHPLC systems and chromatographic columns. Following relative IgG N-glycan quantification in acquired chromatograms, data analysis showed that the most abundant peaks that together made up for three-fourths of the detected IgG N-glycome all had coefficients of variation (CVs) lower than 2%. The highest CVs ranging from 16 to 29% accompanied low abundance glycan peaks with the individual relative peak area below 1% that together made up for <2% of the detected IgG N-glycome. These results show that the tested method is very robust and repeatable, making it suitable for the IgG N-glycan analysis of a large number of samples in a high-throughput manner over a longer period of time.

中文翻译：

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GlycoWorks RapiFluor-MS IgG N-糖链分析在长期高通量糖组学研究中的稳健性和可重复性

蛋白质糖基化是碳水化合物部分与蛋白质骨架的连接，影响蛋白质的结构和功能。糖基化异常与各种疾病有关，甚至在症状出现之前就可以检测到糖基化的一些变化。因此，聚糖已成为引人注目的新生物标志物候选物。存在多种用于小规模聚糖分析的分析方法。然而，对允许大规模糖谱分析的高度稳健和可重复的高通量技术的需求日益增长。在这里，我们描述了使用 GlycoWorks RapiFluor-MS N对免疫球蛋白 G (IgG) N -聚糖分析的稳定性和可重复性的评估-聚糖试剂盒，然后是亲水相互作用超高效液相色谱 (HILIC-UHPLC)，来自 335 个人血浆技术复制品，随机分布在 67 个 96 孔板上。使用多个 UHPLC 系统和色谱柱在 5 个月内收集数据。在获得的色谱图中进行相对 IgG N-糖基定量后，数据分析显示，占检测到的 IgG N-糖组四分之三的最丰富峰的变异系​​数 (CV) 均低于 2%。从 16% 到 29% 的最高 CV 伴随着低丰度的糖链峰，单个相对峰面积低于 1%，它们加起来占检测到的 IgG N的 <2%-糖组。这些结果表明，所测试的方法非常稳健且可重复，使其适用于在较长时间内以高通量方式对大量样品进行IgG N-聚糖分析。