Intoh Atsushi, Kurisaki Akira, Fukuda Hiroyuki, Asashima Makoto
Department of Life Sciences Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Biomed Chromatogr. 2009 Jun;23(6):607-14. doi: 10.1002/bmc.1159.
Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC-HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC-HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC-HILIC has better peptide fractionation ability. We further demonstrated that ZIC-HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC-HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses.
全面的蛋白质组学分析需要高效分离肽混合物，以便随后通过质谱（MS）鉴定蛋白质。然而，从细胞和组织中提取的蛋白质消化后常常产生复杂的肽混合物，这使得直接进行全面的MS分析变得困难。本研究调查了一种用于肽分离步骤的两性离子亲水相互作用液相色谱（ZIC-HILIC）技术，并通过随后的MS分析进行了验证。人血清白蛋白（HSA）是用于此分析的模型蛋白。在进行MS分析以鉴定肽之前，用胰蛋白酶消化HSA，并通过ZIC-HILIC或传统的强阳离子交换（SCX）进行分离。与SCX色谱法相比，使用ZIC-HILIC进行分离显著提高了HSA肽的鉴定率。对鉴定出的肽进行的详细分析表明，ZIC-HILIC具有更好的肽分级分离能力。我们进一步证明，ZIC-HILIC可用于定量检测未分化胚胎干细胞中特异性表达的细胞表面标志物。这些结果表明，ZIC-HILIC作为一种用于全面和定量蛋白质组学分析的新型高效分离方法具有重要价值。
