title: Protocol for Standardizing High-to-Moderate Abundance Protein Biomarker Assessments Through an MRM-with-Standard-Peptides Quantitative Approach.
abstract: Quantitative mass spectrometry (MS)-based approaches are emerging as a core technology for addressing health-related queries in systems biology and in the biomedical and clinical fields. In several 'omics disciplines (proteomics included), an approach centered on selected or multiple reaction monitoring (SRM or MRM)-MS with stable isotope-labeled standards (SIS), at the protein or peptide level, has emerged as the most precise technique for quantifying and screening putative analytes in biological samples. To enable the widespread use of MRM-based protein quantitation for disease biomarker assessment studies and its ultimate acceptance for clinical analysis, the technique must be standardized to facilitate precise and accurate protein quantitation. To that end, we have developed a number of kits for assessing method/platform performance, as well as for screening proposed candidate protein biomarkers in various human biofluids. Collectively, these kits utilize a bottom-up LC-MS methodology with SIS peptides as internal standards and quantify proteins using regression analysis of standard curves. This chapter details the methodology used to quantify 192 plasma proteins of high-to-moderate abundance (covers a 6 order of magnitude range from 31 mg/mL for albumin to 18 ng/mL for peroxidredoxin-2), and a 21-protein subset thereof. We also describe the application of this method to patient samples for biomarker discovery and verification studies. Additionally, we introduce our recently developed Qualis-SIS software, which is used to expedite the analysis and assessment of protein quantitation data in control and patient samples.
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC, V8Z 7X8, Canada.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Percy
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC, V8Z 7X8, Canada.
authors.forename: Juncong
authors.initials: J
authors.lastname: Yang
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC, V8Z 7X8, Canada.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Chambers
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC, V8Z 7X8, Canada.|Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333 ZA, Leiden, Netherlands.
authors.forename: Yassene
authors.initials: Y
authors.lastname: Mohammed
authors.affiliation: AstraZeneca R&D, Innovative Medicines, S-431 83, Mölndal, Sweden.
authors.forename: Tasso
authors.initials: T
authors.lastname: Miliotis
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC, V8Z 7X8, Canada. christoph@proteincentre.com.|Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC, V8P 5C2, Canada. christoph@proteincentre.com.
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
mesh_terms: D000465:Algorithms; D015415:Biomarkers / Q000032:analysis; D002138:Calibration; D019295:Computational Biology / Q000379:methods* / Q000592:standards; D057225:Data Mining / Q000379:methods* / Q000592:standards; D030562:Databases, Protein; D057166:High-Throughput Screening Assays; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards; D011237:Predictive Value of Tests; D011506:Proteins / Q000032:analysis*; D020543:Proteome; D040901:Proteomics / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D012984:Software
chemical_list: D015415:Biomarkers; D011506:Proteins; D020543:Proteome
keywords: Biomarker; Internal standards; MRM; Plasma; Proteomics; Quantitation; Standardization