Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization.
Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization.
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DOI：
10.1007/s00216-010-3991-2
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发表时间：
2010-09
影响因子：
4.3
通讯作者：
Chu, Ivan K.
中科院分区：
文献类型：
--
作者：
Lam, Maggie P. Y.;Siu, S. O.;Lau, Edward;Mao, Xiuli;Sun, H. Z.;Chiu, Philip C. N.;Yeung, William S. B.;Cox, David M.;Chu, Ivan K.
我们开发了一种在微流方案中在线耦合反相（RP）和亲水相互作用液相色谱（HILIC）的新系统。在这种方法中，RP和HILIC之间固有的溶剂不相容性通过使用恒压在线溶剂混合来克服，这使得我们的系统能够有效地分离亲水性和疏水性化合物，用于基于质谱的蛋白质组学应用。当分析牛血清白蛋白、核糖核酸酶B和辣根过氧化物酶的胰蛋白酶消化时，我们观察到，当使用在线RP-HILIC时，肽和糖肽的覆盖范围几乎相同，只有一个样品注射事件-正如我们从两个单独的RP和HILIC分析中所做的那样。偶联系统还能够同时表征一次注射事件中去糖基化糖蛋白的肽和聚糖部分，例如，我们通过检测火鸡卵白蛋白的23种新型聚糖证实了这一点。最后，我们验证了RP-HILIC用于分析高度复杂的生物样品（小鼠软骨细胞裂解液，去糖基化的人血清）的适用性。在线RP-HILIC的覆盖范围和效率的提高使其成为一种全面分离具有显著不同疏水性的成分的可行技术，如肽、糖肽和聚糖。本文的在线版本（doi:10.1007/s00216-010-3991-2）包含补充材料，可供授权用户使用。
We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC—with only a single sample injection event—as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans. The online version of this article (doi:10.1007/s00216-010-3991-2) contains supplementary material, which is available to authorized users.
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