Weng Yejing, Qu Yanyan, Jiang Hao, Wu Qi, Zhang Lihua, Yuan Huiming, Zhou Yuan, Zhang Xiaodan, Zhang Yukui
National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; University of the Chinese Academy of Sciences, Beijing 100039, China.
National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
Anal Chim Acta. 2014 Jun 23;833:1-8. doi: 10.1016/j.aca.2014.04.037. Epub 2014 Apr 26.
Relative quantification of N-glycoproteomes shows great promise for the discovery of candidate biomarkers and therapeutic targets. The traditional protocol for quantitative analysis of glycoproteomes is usually off-line performed, and suffers from long sample preparation time, and the risk of sample loss or contamination due to manual manipulation. In this study, a novel integrated sample preparation platform for quantitative N-glycoproteome analysis was established, with combination of online N-glycopeptide capture by a HILIC column, sample buffer exchange by a N2-assisted HILIC-RPLC interface, deglycosylation by a hydrophilic PNGase F immobilized enzymatic reactor (hIMER) and solid dimethyl labeling on a C18 precolumn. To evaluate the performance of such a platform, two equal aliquots of immunoglobulin G (IgG) digests were sequentially pretreated, followed by MALDI-TOF MS analysis. The signal intensity ratio of heavy/light (H/L) labeled deglycosylated peptides with the equal aliquots was 1.00 (RSD=6.2%, n=3), much better than those obtained by the offline protocol, with H/L ratio as 0.76 (RSD=11.6%, n=3). Additionally, the total on-line sample preparation time was greatly shortened to 160 min, much faster than that of offline approach (24h). Furthermore, such an integrated pretreatment platform was successfully applied to analyze the two kinds of hepatocarcinoma ascites syngeneic cell lines with high (Hca-F) and low (Hca-P) lymph node metastasis rates. For H/L labeled Hca-P lysates with the equal aliquots, 99.6% of log2 ratios (H/L) of quantified glycopeptides ranged from -1 to 1, demonstrating high accuracy of the developed sample preparation strategy. By triplicated analysis of glycopeptides and non-glycopeptides of Hca-F and Hca-P lysates, 43 up-regulated and 30 down-regulated (Hca-F/P) N-glycosylation sites, and 11 significantly changed N-glycoproteins were successfully quantified, and most of them were related to tumorigenesis and tumor metastasis. All these results demonstrate the developed integrated N-glycoprotein pretreatment platform is of great power for the accurate, precise and high-throughput analysis of N-glycoproteomes.
N-糖蛋白质组的相对定量分析在发现候选生物标志物和治疗靶点方面显示出巨大潜力。传统的糖蛋白质组定量分析方案通常是离线进行的，存在样品制备时间长以及由于手动操作导致样品损失或污染的风险。在本研究中，建立了一种用于定量N-糖蛋白质组分析的新型集成样品制备平台，该平台结合了亲水相互作用液相色谱（HILIC）柱在线捕获N-糖肽、N2辅助的HILIC-反相液相色谱（RPLC）接口进行样品缓冲液交换、亲水性肽-N-糖苷酶F固定化酶反应器（hIMER）进行去糖基化以及在C18预柱上进行固相二甲基标记。为了评估该平台的性能，对两份等量的免疫球蛋白G（IgG）消化产物进行了顺序预处理，随后进行基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析。等量样品中重/轻（H/L）标记的去糖基化肽的信号强度比为1.00（相对标准偏差RSD = 6.2%，n = 3），远优于离线方案得到的结果，离线方案的H/L比为0.76（RSD = 11.6%，n = 3）。此外，在线样品制备总时间大幅缩短至160分钟，比离线方法（24小时）快得多。此外，这种集成预处理平台成功应用于分析两种具有高（Hca-F）和低（Hca-P）淋巴结转移率的肝癌腹水同基因细胞系。对于等量的H/L标记的Hca-P裂解物，99.6%的定量糖肽的log2比值（H/L）在-1至1之间，表明所开发的样品制备策略具有很高的准确性。通过对Hca-F和Hca-P裂解物的糖肽和非糖肽进行三次重复分析，成功定量了43个上调和30个下调（Hca-F/P）的N-糖基化位点以及11个显著变化的N-糖蛋白，其中大多数与肿瘤发生和肿瘤转移有关。所有这些结果表明，所开发的集成N-糖蛋白预处理平台在N-糖蛋白质组的准确、精确和高通量分析方面具有强大的能力。
