title: Multiplexed MRM with Internal Standards for Cerebrospinal Fluid Candidate Protein Biomarker Quantitation.
abstract: Multiplexed quantitation is essential for discovering, verifying, and validating biomarkers for risk stratification, disease prognostication, and therapeutic monitoring. The most promising strategy for quantifying unverified protein biomarkers in biofluids relies on selected/multiple reaction monitoring (SRM or MRM) technology with isotopically labeled standards employed within a bottom-up proteomic workflow. Since cerebrospinal fluid (CSF) is an important fluid for studying central nervous system (CNS) related diseases, we sought to develop a rapid, antibody- and fractionation-free MRM-based approach with a complex mixture of peptide standards to quantify a highly multiplexed panel of candidate protein biomarkers in human CSF. Development involved peptide transition optimization, denaturation/digestion protocol evaluation, transition interference screening, and protein quantitation via peptide standard curves. The final method exhibited excellent reproducibility (average coefficient of variation of <1% for retention time and <6% for signal) and breadth of quantitation (130 proteins from 311 interference-free peptides) in a single 43-min run. These proteins are of high-to-low abundance with determined concentrations from 118 μg/mL (serum albumin) to 550 pg/mL (apolipoprotein C-I). Overall, the method consists of the most highly multiplexed and broadest panel of candidate protein biomarkers in human CSF reported thus far and is well suited for subsequent verification studies on patient samples.
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, University of Victoria , Vancouver Island Technology Park, 3101-4464 Markham Street, 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, University of Victoria , Vancouver Island Technology Park, 3101-4464 Markham Street, Victoria, BC V8Z 7X8, Canada.
authors.forename: Juncong
authors.initials: J
authors.lastname: Yang
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, University of Victoria , Vancouver Island Technology Park, 3101-4464 Markham Street, 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, University of Victoria , Vancouver Island Technology Park, 3101-4464 Markham Street, Victoria, BC V8Z 7X8, Canada.
authors.forename: Romain
authors.initials: R
authors.lastname: Simon
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, University of Victoria , Vancouver Island Technology Park, 3101-4464 Markham Street, Victoria, BC V8Z 7X8, Canada.
authors.forename: Darryl B
authors.initials: DB
authors.lastname: Hardie
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, University of Victoria , Vancouver Island Technology Park, 3101-4464 Markham Street, Victoria, BC V8Z 7X8, Canada.|Department of Biochemistry and Microbiology, University of Victoria , Petch Building Room 207, 3800 Finnerty Road, Victoria, BC V8P 5C2, Canada.
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
keywords: MRM; cerebrospinal fluid; multiplexed; protein quantitation; robust