Abstract
We proteotyped blood plasma from 30 mouse knockout strains and corresponding wild-type mice from the International Mouse Phenotyping Consortium. We used targeted proteomics with internal standards to quantify 375 proteins in 218 samples. Our results provide insights into the manifested effects of each gene knockout at the plasma proteome level. We first investigated possible contamination by erythrocytes during sample preparation and labeled, in one case, up to 11 differential proteins as erythrocyte originated. Second, we showed that differences in baseline protein abundance between female and male mice were evident in all mice, emphasizing the necessity to include both sexes in basic research, target discovery, and preclinical effect and safety studies. Next, we identified the protein signature of each gene knockout and performed functional analyses for all knockout strains. Further, to demonstrate how proteome analysis identifies the effect of gene deficiency beyond traditional phenotyping tests, we provide in-depth analysis of two strains, C8a−/− and Npc2+/−. The proteins encoded by these genes are well-characterized providing good validation of our method in homozygous and heterozygous knockout mice. Ig alpha chain C region, a poorly characterized protein, was among the differentiating proteins in C8a−/−. In Npc2+/− mice, where histopathology and traditional tests failed to differentiate heterozygous from wild-type mice, our data showed significant difference in various lysosomal storage disease-related proteins. Our results demonstrate how to combine absolute quantitative proteomics with mouse gene knockout strategies to systematically study the effect of protein absence. The approach used here for blood plasma is applicable to all tissue protein extracts.
Highlights
Mus musculus is the most used animal model in scientific research
These studies have revealed functional genomics insights beyond that derived from sequencing alone[17,18,19,20]. Such efforts have included the analysis of cells and tissues from wild type, transgenic, knockin, and knockout strains, and mice labeled in vivo with isotope using mass spectrometrybased methods[21,22,23,24]
Differential expression is inferred from mass spectrum signal intensity and good comparability across groups can be achieved using labeling approaches such as isobaric tagging using Tandem Mass Tag (TMT), Isobaric Tag for Relative and Absolute Quantitation, or Stable Isotope Labeling with Amino acids in Cell culture (SILAC) 27
Summary
Mus musculus is the most used animal model in scientific research. It has high similarity with humans at the molecular level with 99% of human genes having homologs in the mouse genome[1]. Parallel to the various sequencing efforts, comprehensive studies at the proteome level have been performed in recent years and provided insight into the proteins that are differentially expressed between cells, tissues, organs, or organ systems, or are related to a specific condition or disease. To our knowledge, this is the first large-scale ing whether the presence of intracellular proteins in plasma reflects a biological condition, or if they are the result of sample processing.
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