Machtejevas Egidijus, John Harald, Wagner Knut, Ständker Ludger, Marko-Varga György, Forssmann Wolf-Georg, Bischoff Rainer, Unger Klaus K
Institut fuer Anorganische Chemie und Analytische Chemie, Johannes Gutenberg-Universitaet, Duesbergweg 10-14, D-55099 Mainz, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2004 Apr 15;803(1):121-30. doi: 10.1016/j.jchromb.2003.07.015.
A comprehensive on-line sample clean-up with an integrated two-dimensional HPLC system was developed for the analysis of natural peptides. Samples comprised of endogenous peptides with molecular weights up to 20 kDa were generated from human hemofiltrate (HF) obtained from patients with chronic renal failure. The (poly-)peptides were separated using novel silica-based restricted access materials with strong cation-exchange functionalities (SCX-RAM). The size-selective sample fractionation step is followed by cation-exchange chromatography as the first dimension. The subsequent second dimension of separation is based on hydrophobic interaction using four parallel short reversed-phase (RP) columns implemented via a fully automated column switching technique. More than 1000 peaks were resolved within the total analysis time of 96 min. Substances of selected peaks were sampled to analyse their molecular weights by off-line MALDI-TOF mass spectrometry and to determine their amino acid sequence by Edman degradation. The potential for comprehensive peptide mapping and identification is demonstrated.
开发了一种用于分析天然肽的综合在线样品净化与集成二维高效液相色谱系统。从慢性肾衰竭患者的人血液滤过液（HF）中生成了分子量高达20 kDa的内源性肽组成的样品。使用具有强阳离子交换功能的新型硅胶基受限进样材料（SCX-RAM）分离（多）肽。尺寸选择性样品分级步骤之后是作为第一维的阳离子交换色谱。随后的第二维分离基于使用通过全自动柱切换技术实现的四个平行短反相（RP）柱的疏水相互作用。在96分钟的总分析时间内分离出1000多个峰。对选定峰的物质进行离线基质辅助激光解吸电离飞行时间质谱分析其分子量，并通过埃德曼降解法确定其氨基酸序列。证明了全面肽图谱分析和鉴定的潜力。
