Multiplexed MRM‐based assays for the quantitation of proteins in mouse plasma and heart tissue

Abstract

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.

Concentration Determination of >200 Proteins in Dried Blood Spots for Biomarker Discovery and Validation

“…After preanalytical sample preparation (described above) of two replicate DBS samples, the samples were subjected to solid phase extraction (25) and lyophilisation. Samples were solubilized in 210 l of 3% aqueous acetonitrile in water and used for LC-MS/MS on an Orbitrap Fusion Tribrid coupled to an EASYnLC 1000 HPLC system via a Nanospray Flex NG source (Thermo Fisher Scientific), as previously described (26,27). The only modification made was to inject 1 l of each technical replicate, which was equivalent to ϳ 1 g of total protein.…”

“…Response Curve-CPTAC experiment 1 (20) was used as a guide to assess the lower limit of quantification (LLOQ) for each target peptide. Using SIS peptides to spike trypsin digested DBS (DBS matrix), the reverse-curve (26,27) approach was used to generate an eight-point response curve (in addition to a blank), to determine the LLOQ. The 336 SIS peptides to be tested were used to make working solutions of SIS peptides containing 5000 fmol/l of peptide in 3% ACN/H 2 O.…”

“…Multiday Validation of Assay Analytical Repeatability-For peptides for which an LLOQ was determined, based on the criteria defined above, a multi-day assessment of repeatability was performed, using CPTAC experiment 2 (20) as a guide. Repeatability was assessed using the reverse-curve approach (26,27), which involves varying the SIS concentration and using NAT and/or endogenous proteolytic peptides as normalizers, or by using the SIS peptide without normalization, for targets for which the NAT peptide was not available or where the endogenous proteolytic peptide was contributing to the variability. DBS matrix spiked with NAT peptides was used for the blank data points which would later be used to assess the limit of detection, defined as three standard deviations higher than the signal measured across the blank samples (three replicates).…”

Current trends in quantitative proteomics – an update

“…Although its collection is even more invasive than plasma, cerebrospinal fluid may still be the best option for biomarkers of brain‐related diseases . Multiplexed MRM assays for animal models of human diseases, such as the mouse, have also been developed for use in medical research …”

The development of SRM assays is transforming proteomics research

“…Whereas proteome-wide SRM assays have recently been developed for a small number of species, this is not so for the mouse. In this issue, Percy et al [1] report the development of hundreds of mouse SRM assays. Their development required shotgun MS to identify proteotypic peptides, synthesis, and LC-MS characterization of peptide standards, and interlaboratory SRM to robustly assess the quality of the assays.…”