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Profiling of endogenous peptides by multidimensional liquid chromatography: On‐line automated sample cleanup for biomarker discovery in human urine
Author(s) -
Machtejevas Egidijus,
MarkoVarga Gyorgy,
Lindberg Claes,
Lubda Dieter,
Hendriks Robertus,
Unger Klaus K.
Publication year - 2009
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200900058
Subject(s) - chromatography , chemistry , biomarker discovery , repeatability , sample preparation , adsorption , reproducibility , ion suppression in liquid chromatography–mass spectrometry , dilution , biological fluids , elution , urine , analytical chemistry (journal) , mass spectrometry , liquid chromatography–mass spectrometry , proteomics , biochemistry , physics , organic chemistry , gene , thermodynamics
Abstract 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).