Hustoft Hanne Kolsrud, Brandtzaeg Ole Kristian, Rogeberg Magnus, Misaghian Dorna, Torsetnes Silje Bøen, Greibrokk Tyge, Reubsaet Léon, Wilson Steven Ray, Lundanes Elsa
Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway.
1] Department of Chemistry, University of Oslo, Post Box 1033 Blindern, NO-0315 Oslo, Norway [2] Department of Neurology, Akershus University Hospital, 1478 Lørenskog, Norway.
Sci Rep. 2013 Dec 16;3:3511. doi: 10.1038/srep03511.
Reliable, sensitive and automatable analytical methodology is of great value in e.g. cancer diagnostics. In this context, an on-line system for enzymatic cleavage of proteins, subsequent peptide separation by liquid chromatography (LC) with mass spectrometric detection has been developed using "sub-chip" columns (10-20 μm inner diameter, ID). The system could detect attomole amounts of isolated cancer biomarker progastrin-releasing peptide (ProGRP), in a more automatable fashion compared to previous methods. The workflow combines protein digestion using an 20 μm ID immobilized trypsin reactor with a polymeric layer of 2-hydroxyethyl methacrylate-vinyl azlactone (HEMA-VDM), desalting on a polystyrene-divinylbenzene (PS-DVB) monolithic trap column, and subsequent separation of resulting peptides on a 10 μm ID (PS-DVB) porous layer open tubular (PLOT) column. The high resolution of the PLOT columns was maintained in the on-line system, resulting in narrow chromatographic peaks of 3-5 seconds. The trypsin reactors provided repeatable performance and were compatible with long-term storage.
可靠、灵敏且可自动化的分析方法在例如癌症诊断等领域具有重要价值。在此背景下，已开发出一种在线系统，用于蛋白质的酶切，随后通过液相色谱（LC）结合质谱检测进行肽段分离，该系统使用了“亚芯片”柱（内径10 - 20 μm）。与先前方法相比，该系统能够以更自动化的方式检测到阿托摩尔量的分离出的癌症生物标志物胃泌素释放肽前体（ProGRP）。该工作流程将使用内径20 μm的固定化胰蛋白酶反应器（带有甲基丙烯酸2 - 羟乙酯 - 乙烯基氮杂环丁烷（HEMA - VDM）聚合物层）进行蛋白质消化、在聚苯乙烯 - 二乙烯基苯（PS - DVB）整体捕集柱上进行脱盐以及随后在内径10 μm的（PS - DVB）多孔层开口管柱（PLOT柱）上分离所得肽段相结合。PLOT柱的高分辨率在在线系统中得以保持，产生了3 - 5秒的窄色谱峰。胰蛋白酶反应器具有可重复的性能，并且与长期储存兼容。
