Multiplexed MRM-based assays for the quantitation of proteins in mouse plasma and heart tissue.
The mouse is the most commonly used laboratory animal, with more than 14 million mice being used for research each year in North America alone. The number and diversity of mouse models is increasing rapidly through genetic engineering strategies, but detailed characterization of these models is still challenging because most phenotypic information is derived from time-consuming histological and biochemical analyses. To expand the biochemists' toolkit, we generated a set of targeted proteomic assays for mouse plasma and heart tissue, utilizing bottom-up LC/MRM-MS with isotope-labeled peptides as internal standards. Protein quantitation was performed using reverse standard curves, with LC-MS platform and curve performance evaluated by quality control standards. The assays comprising the final panel (101 peptides for 81 proteins in plasma; 227 peptides for 159 proteins in heart tissue) have been rigorously developed under a fit-for-purpose approach and utilize stable-isotope labeled peptides for every analyte to provide high-quality, precise relative quantitation. In addition, the peptides have been tested to be interference-free and the assay is highly multiplexed, with reproducibly determined protein concentrations spanning >4 orders of magnitude. The developed assays have been used in a small pilot study to demonstrate their application to molecular phenotyping or biomarker discovery/verification studies.
University of Victoria-Genome British Columbia Proteomics Centre, , Vancouver Island Technology Park, Victoria, BC, Canada.
Andrew J
AJ
Percy
MRM Proteomics, , Vancouver Island Technology Park, Victoria, BC, Canada.
Sarah A
SA
Michaud
Institute of Parasitology, McGill University, Montreal, QC, Canada.
Armando
A
Jardim
University of Victoria-Genome British Columbia Proteomics Centre, , Vancouver Island Technology Park, Victoria, BC, Canada.
Nicholas J
NJ
Sinclair
MRM Proteomics, , Vancouver Island Technology Park, Victoria, BC, Canada.
Suping
S
Zhang
University of Victoria-Genome British Columbia Proteomics Centre, , Vancouver Island Technology Park, Victoria, BC, Canada.|Center for Proteomics and Metabolomics, Leiden University Medical Center, ZA, Leiden, Netherlands.
Yassene
Y
Mohammed
MRM Proteomics, , Vancouver Island Technology Park, Victoria, BC, Canada.
Andrea L
AL
Palmer
University of Victoria-Genome British Columbia Proteomics Centre, , Vancouver Island Technology Park, Victoria, BC, Canada.
Darryl B
DB
Hardie
University of Victoria-Genome British Columbia Proteomics Centre, , Vancouver Island Technology Park, Victoria, BC, Canada.
Juncong
J
Yang
University of Victoria-Genome British Columbia Proteomics Centre, , Vancouver Island Technology Park, Victoria, BC, Canada.
Andre M
AM
LeBlanc
University of Victoria-Genome British Columbia Proteomics Centre, , Vancouver Island Technology Park, Victoria, BC, Canada.|Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada.
Christoph H
CH
Borchers
D000818:Animals; D015415:Biomarkers / Q000097:blood; D001798:Blood Proteins / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*
D015415:Biomarkers; D001798:Blood Proteins
Animal proteomics; Biomarker; Heart; MRM; Molecular phenotyping; Mouse models; Plasma; Quantitative proteomics; Tissue