An Automated RP–SCX Solid-Phase Extraction Procedure for Urinary Peptidomics Biomarker Discovery Studies
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.
Key wordsUrine Peptidomics RP–SCX purification Native peptides
This study was supported by the European Union, Sixth framework program, Specific Measures in support of International Cooperation (INCO), contract 517733.
- 2.Adam BL, Qu Y, Davis JW, Ward MD, Clements MA, Cazares LH, Semmes OJ, Schellhammer PF, Yasui Y, Feng Z, Wright GL Jr (2002) Serum protein fingerprinting coupled with a pattern-matching algorithm distinguishes prostate cancer from benign prostate hyperplasia and healthy men. Cancer Res 62:3609–3614PubMedGoogle Scholar
- 24.Hughes NC, Wong EY, Fan J, Bajaj N (2007) Determination of carryover and contamination for mass spectrometry-based chromatographic assays. The American Association of Pharmaceutical Scientists journal 9:E353–E360Google Scholar