An automated RP-SCX solid-phase extraction procedure for urinary peptidomics biomarker discovery studies.

ABSTRACT

Urine represents the most easily obtainable body fluid and consequently one of the most common samples in clinical chemistry. The majority of pathological changes in human organs may well be reflected in urine. In this way, urine analysis can aid in disease diagnosis, treatment monitoring, and prognosis. Currently, the most commonly used method for identification of new urine biomarkers involves centrifugation of the urine sample to collect either the soluble urine proteins or the urinary exosomes followed by 1 or 2 protein purification and separation steps before visualization and finally identification of potential biomarkers, usually by mass spectrometry. Here we present a generally applicable, rapid, and robust method for screening large number of urine samples, resulting in a broad spectrum of native peptides, as a tool to be used for biomarker discovery. The method combines online sample pretreatment with a well-established mass spectrometric technique. Native peptides are extracted from urine samples on a miniaturized reverse-phase-strong cation exchange cartridge system. As the proper identification of native peptides often requires combination of data acquired on different mass analyzers, we have aimed at a procedure providing us with sufficient material to identify and characterize the differentially expressed markers.