title: A standardized and reproducible urine preparation protocol for cancer biomarkers discovery.
abstract: A suitable and standardized protein purification technique is essential to maintain consistency and to allow data comparison between proteomic studies for urine biomarker discovery. Ultimately, efforts should be made to standardize urine preparation protocols. The aim of this study was to develop an optimal analytical protocol to achieve maximal protein yield and to ensure that this method was applicable to examine urine protein patterns that distinguish disease and disease-free states. In this pilot study, we compared seven different urine sample preparation methods to remove salts, and to precipitate and isolate urinary proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles showed that the sequential preparation of urinary proteins by combining acetone and trichloroacetic acid (TCA) alongside high speed centrifugation (HSC) provided the best separation, and retained the most urinary proteins. Therefore, this approach is the preferred method for all further urine protein analysis.
authors.affiliation: Cancer Care Centre, St George Hospital, Gray St, Kogarah, NSW, Australia. ; St George and Sutherland Clinical School, Faculty of Medicine, University of New South Wales (UNSW), Kensington, NSW, Australia. ; SEALS, Anatomical Pathology, St George Hospital, Gray St, Kogarah, NSW, Australia.
authors.forename: Julia
authors.initials: J
authors.lastname: Beretov
authors.affiliation: Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, University of New South Wales (UNSW), Kensington, NSW, Australia.
authors.forename: Valerie C
authors.initials: VC
authors.lastname: Wasinger
authors.affiliation: Breast Surgery, St George Private Hospital, South St, Kogarah, NSW, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Schwartz
authors.affiliation: Cancer Care Centre, St George Hospital, Gray St, Kogarah, NSW, Australia. ; St George and Sutherland Clinical School, Faculty of Medicine, University of New South Wales (UNSW), Kensington, NSW, Australia.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Graham
authors.affiliation: Cancer Care Centre, St George Hospital, Gray St, Kogarah, NSW, Australia. ; St George and Sutherland Clinical School, Faculty of Medicine, University of New South Wales (UNSW), Kensington, NSW, Australia.
authors.forename: Yong
authors.initials: Y
authors.lastname: Li
keywords: LC-MS/MS; SDS-PAGE; biomarker; breast cancer; proteomics; urine