Department of Chemistry, The University of Hong Kong, Hong Kong, PR China.
Electrophoresis. 2011 Nov;32(21):2930-40. doi: 10.1002/elps.201100092. Epub 2011 Oct 18.
In this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected.
本文描述了一种在线反相/反相（RP-RP）和多孔石墨化碳（PGC）液相色谱（LC）联用方法，用于蛋白质组学和糖蛋白质组学样品的多组分分析。该系统的在线 RP-RP 部分在 pH 2 和 10 下基于序列疏水性提供全面的二维肽分离。未被 RP 保留的亲水性成分（如聚糖、糖肽）自动被引导到 PGC 柱进行进一步的捕获和分离。此外，RP-RP/PGC 系统还可以同时扩展亲水性范围和分析的峰容量。使用 11 种蛋白质混合物，我们发现该系统可以有效地从单个样品中分离天然肽和释放的 N-聚糖。我们通过使用人绒毛膜癌细胞系（BeWo）的 25 μg 裂解物来评估该系统对复杂生物样品分析的适用性，从单个实验中自信地鉴定出 1449 种蛋白质，从技术重复中鉴定出多达 1909 种不同的蛋白质。PGC 馏分通过包括额外的亲水序列来增加序列覆盖率，这些序列占 BeWo 裂解物中总鉴定肽的 6.9%，对亲水基序和蛋白质的检测有明显的偏好。此外，RP-RP/PGC 适用于复杂糖组学样品的分析，如我们对人血清中凝集素 A 提取的糖蛋白质组的分析所示；总共同时检测到 134 种潜在的 N-糖基化血清蛋白、151 个可能的 N-糖基化位点和 40 多种可能的凝集素 A 识别的 N-聚糖结构。
