Profiling of endogenous peptides by multidimensional liquid chromatography: On-line automated sample cleanup for biomarker discovery in human urine
Profiling of endogenous peptides by multidimensional liquid chromatography: On-line automated sample cleanup for biomarker discovery in human urine
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DOI：
10.1002/jssc.200900058
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发表时间：
2009-07-01
影响因子：
3.1
通讯作者：
Unger, Klaus K.
中科院分区：
文献类型：
--
作者：
Machtejevas, Egidijus;Marko-Varga, Gyorgy;Unger, Klaus K.
设计了一种简单灵活的系统，采用柱切换技术，通过同时进行样品净化和痕量富集柱上直接进样，实现了对过滤后尿样中相对分子质量小于15 kDa的多肽和蛋白质的分析。样品中带正电的多肽和小蛋白质被吸引到RAM-SCX柱的内部带负电的孔结构，而较大的蛋白质和未带电或带负电的化合物被排除在外。用生物样品进行预处理后，可以大量注入样品。描述和讨论了正确使用RAM-SCX色谱柱的几个重要的可调参数。主要参数是：i)柱对样品过载很敏感，这可能会导致多肽的吸附发生剧烈变化；ii)吸附看起来与流速和浓度有关，因为样品分子需要时间穿透内部孔结构以找到互补的定向吸附位置；iii)样品在加载过程中的稀释和pH调节。利用人体尿样验证了该分离平台的生物相容性和原理。提供了关于重复性以及不同合成批次的限制访问材料(RAM)的再现性的数据。
A simple and flexible system, employing a column switching technique, has been designed to allow the analysis of peptides and proteins smaller than 15 kDa by molecular weight in filtered urine samples by performing a direct on-column injection utilising simultaneous sample clean-up and trace enrichment. The positively charged peptides and small proteins in the sample are attracted to the inner, negatively charged pore structure of the RAM-SCX column while the larger proteins and uncharged or negatively charged compounds are excluded. After preconditioning with the biological sample, large amounts of sample can be injected. Several important and adjustable parameters for the proper use of a RAM-SCX column are described and discussed. The main parameters being: i) the column is sensitive to sample overloading, which may result in drastic changes in the adsorption of peptides; ii) adsorption appears to be flow-rate and concentration dependent, as the sample molecules need time to penetrate into the internal pore structure in order to find complimentary orientated adsorption sites; iii) dilution and pH adjustment Of sample during the loading process. The biocompatibility and proof-of-principle of this separation platform was demonstrated using human urine samples. Data are presented on repeatability as well as on the reproducibility of different synthesised batches of restricted access material (RAM).