Development, Validation, and Application of a MRM with SIS Peptide Strategy to Quantify Multiplexed Panels of Proteins in Mouse Plasma and Heart Tissues
- Organism: Mus musculus
- Instrument: Agilent’s 6490 Triple Quadrupole
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 the advancement of new genetic engineering strategies; however, detailed characterization of these models is still challenging because most phenotypic information is derived from time-consuming histological and biochemical analyses. To expedite molecular phenotyping, we have developed a series of MRM assays to quantify candidate protein biomarkers in mouse plasma and heart tissue. The method utilizes bottom-up LC-MS/MS with isotopically labeled peptides serving as internal standards. Quantitation was achieved by reverse standard curves, while LC-MS platform and curve performance was evaluated with quality control (QC) standards. The mouse protein assays (81 for plasma, 159 for heart) were validated under a fit-for-purpose approach then applied to experimental mouse tissue samples to demonstrate their utility in molecular phenotyping or screening studies. These tier 2 research assays have the necessary robustness for routine biomarker assessment of mouse models. Such studies will help provide insight into the tissue-specific cellular processes and pathways affected in disease, and help enhance and expedite the breeding of new mouse models.
Unique proteotypic tryptic peptides were chosen as surrogates for mouse proteins then synthesized, purified, and characterized in their isotopically labeled state. After optimizing peptide- and transition-specific parameters, a smaller panel of targets was evaluated against various methodological variations using mouse plasma and heart tissues (both supplied from Bioreclamation). The final method involves simple sample pre-treatment (utilizes urea for protein denaturation) prior to overnight tryptic digestion, SIS peptide addition, and UHPLC-MRM/MS on an Agilent 6490 triple quadrupole. Interference testing and method reproducibility were conducted, with protein quantitation being performed in replicate analyses using standard reverse curves from a panel of qualified proteotypic peptides. The validated results were applied to mouse plasma and heart tissue samples to demonstrate the methods utility in molecular phenotyping or screening studies.
Using an MRM-with-SIS-peptide approach, a set of robust assays for quantifying large numbers of candidate protein biomarkers in mouse plasma and heart tissue has been developed. The validated panel consists of 81 proteins (represented by 101 peptides) for mouse plasma and 159 proteins (represented by 227 peptides) for heart tissue. These were reproducibly quantified (inter-assay CVs <15%, on average), with protein concentrations spanning >4 orders of magnitude.
Created on 5/20/16 11:30 AM