title: Precise quantitation of 136 urinary proteins by LC/MRM-MS using stable isotope labeled peptides as internal standards for biomarker discovery and/or verification studies.
abstract: Spurred on by the growing demand for panels of validated disease biomarkers, increasing efforts have focused on advancing qualitative and quantitative tools for more highly multiplexed and sensitive analyses of a multitude of analytes in various human biofluids. In quantitative proteomics, evolving strategies involve the use of the targeted multiple reaction monitoring (MRM) mode of mass spectrometry (MS) with stable isotope-labeled standards (SIS) used for internal normalization. Using that preferred approach with non-invasive urine samples, we have systematically advanced and rigorously assessed the methodology toward the precise quantitation of the largest, multiplexed panel of candidate protein biomarkers in human urine to date. The concentrations of the 136 proteins span >5 orders of magnitude (from 8.6 μg/mL to 25 pg/mL), with average CVs of 8.6% over process triplicate. Detailed here is our quantitative method, the analysis strategy, a feasibility application to prostate cancer samples, and a discussion of the utility of this method in translational studies.
authors: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada. Electronic address: ajpercy@proteincentre.com.
authors: Andrew J
authors: AJ
authors: Percy
authors: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada.
authors: Juncong
authors: J
authors: Yang
authors: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada.
authors: Darryl B
authors: DB
authors: Hardie
authors: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada.
authors: Andrew G
authors: AG
authors: Chambers
authors: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada.
authors: Jessica
authors: J
authors: Tamura-Wells
authors: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada; Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada. Electronic address: christoph@proteincentre.com.
authors: Christoph H
authors: CH
authors: Borchers
mesh_terms: D000368:Aged; D014408:Biomarkers, Tumor / Q000652:urine*; D002250:Carbon Radioisotopes; D002853:Chromatography, Liquid / Q000379:methods* / Q000592:standards; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards; D009363:Neoplasm Proteins / Q000652:urine*; D009590:Nitrogen Radioisotopes; D010455:Peptides / Q000592:standards; D011471:Prostatic Neoplasms / Q000175:diagnosis; D040901:Proteomics / Q000379:methods; D012015:Reference Standards; D016482:Urinalysis / Q000379:methods
chemical_list: D014408:Biomarkers, Tumor; D002250:Carbon Radioisotopes; D009363:Neoplasm Proteins; D009590:Nitrogen Radioisotopes; D010455:Peptides
keywords: Internal standard; MRM; Multiplexed; Peptide; Protein quantitation; Urine