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. 2018 Jun 27:1:78.
doi: 10.1038/s42003-018-0087-6. eCollection 2018.

Molecular phenotyping of laboratory mouse strains using 500 multiple reaction monitoring mass spectrometry plasma assays

Sarah A Michaud  1 , Nicholas J Sinclair  1 , Helena Pětrošová  1 , Andrea L Palmer  1 , Adam J Pistawka  1 , Suping Zhang  1 , Darryl B Hardie  1 , Yassene Mohammed  1   2 , Azad Eshghi  1 , Vincent R Richard  3 , Albert Sickmann  4 , Christoph H Borchers  5   6   7   8
Affiliations

Molecular phenotyping of laboratory mouse strains using 500 multiple reaction monitoring mass spectrometry plasma assays

Sarah A Michaud et al. Commun Biol. .

Abstract

Mouse is the predominant experimental model for the study of human disease due, in part, to phylogenetic relationship, ease of breeding, and the availability of molecular tools for genetic manipulation. Advances in genome-editing methodologies, such as CRISPR-Cas9, enable the rapid production of new transgenic mouse strains, necessitating complementary high-throughput and systematic phenotyping technologies. In contrast to traditional protein phenotyping techniques, multiple reaction monitoring (MRM) mass spectrometry can be highly multiplexed without forgoing specificity or quantitative precision. Here we present MRM assays for the quantitation of 500 proteins and subsequently determine reference concentration values for plasma proteins across five laboratory mouse strains that are typically used in biomedical research, revealing inter-strain and intra-strain phenotypic differences. These 500 MRM assays will have a broad range of research applications including high-throughput phenotypic validation of novel transgenic mice, identification of candidate biomarkers, and general research applications requiring multiplexed and precise protein quantification.

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Conflict of interest statement

C.H.B. is the CSO of MRM Proteomics, Inc. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Repeatability and dynamic range of designed assays (CPTAC Experiment 2). a Total variability of peak areas, as calculated from the intra-assay and inter-assay CoVs across five independent experiments. Box and whisker plots were generated using the Tukey method; mean (+) and outliers (circles) are shown. Blue, white, and red boxes correspond to the total variability at low (2.5× or 5× assay LLOQ), medium (50× assay LLOQ) and high (500× assay LLOQ) concentrations of the spiked-in SIS peptide, respectively. The dotted line represents the 20% cut-off, as defined by CPTAC. b The dynamic range of the designed assays. Assay LLOQ (blue line), assay ULOQ (red line), and the detectable concentrations of the respective endogenous analyte (black dots, N = 367) in pooled plasma of C57BL/6 mice (N = 30) are shown
Fig. 2
Fig. 2
Stability of designed assays (CPTAC Experiment 4). a Intra-assay variability in SIS/NAT peak area ratio. Triplicate aliquots of the samples were analyzed by duplicate LC/MRM-mass spectrometry injections after 0, 6, or 24 h on the autosampler (4 °C). Additional aliquots were frozen at −80 °C, and analyzed identically after thawing (T−1×), after a second freeze-thaw cycle (T−2×), or after 4 weeks at −80 °C (T−4w). b Inter-assay variability in SIS/NAT peak area ratios comparing each of the six injections across all time points and freeze-thaw cycles. Box and whisker plots were generated using the Tukey method; mean (+) and outliers (circles) are shown. The dotted line represents the 20% cut-off, as defined by CPTAC. c SIS/NAT peak area ratios of 20 representative peptides across six experimental time points. T, freeze-thaw; w, weeks; Inj, injection
Fig. 3
Fig. 3
Plasma protein profiles of five laboratory mouse strains. a Heat map presenting plasma protein abundances (log-transformed) in each mouse strain (male and female protein abundance were combined). b Frequency distribution of the slope and R2 values calculated from 91 distinct linear regression analyses of plasma protein abundance (log transformed) across all male and female mouse strains and colonies (5 strains, 7 colonies, male and female separated, resulting in 91 distinct linear regression curves). c Intra-strain comparison of the abundance of plasma proteins in C57BL/6 mice obtained from three different vendors (y axis is in antilog scale). d Frequency distribution of percent differences in proteins in C57BL/6 mice from three different vendors. All differences in peptide abundance are shown, including those without statistical significance. e Intra-strain variability of protein abundance in plasma of C57BL/6 mice. Only proteins whose abundances differed between at least two C57BL/6 strains are shown (p < 0.05 by two-way ANOVA with Tukey’s correction for multiple comparisons). f Strain-specific abundance of immunoglobulins (Ig) in plasma. Abundances are shown only for target analytes detected within the dynamic range of the respective assays in plasma from at least four individual mice for each mouse strain; grey coloring indicates analytes that were not detected or were not quantifiable in plasma of the respective mouse strains. N = six mice/strain (three male and three female). Means of log-transformed data are shown; concentrations are those of the surrogate peptides (pmol mL−1 of plasma). CRL, Charles River Laboratories; J, Jackson Laboratory; and BR, BioReclamationIVT
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