Proteotyping of knockout mouse strains reveals sex- and strain-specific signatures in blood plasma

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.

Proteomic Evolution from Acute to Post-COVID-19 Conditions

“…The sample digest preparation protocol was developed previously and was optimized and used in multiple follow-up studies. ,,− In our current work, we used a urea-based protocol in which 10 μL of plasma was diluted with 20 μL of 9 M urea/20 mM dithiothreitol and incubated for 30 min at 37 °C to achieve denaturation and reduction. The samples were alkylated with iodoacetamide (40 mM final concentration) for 30 min at room temperature in the dark, and then, the samples were diluted 10-fold in 100 mM Tris prior to tryptic digestion.…”

Importance of fatty acid binding proteins in cellular function and organismal metabolism

“…sex, genetic background, nutrient status) is an important modifier of the phenotypic outcomes of Fabp gene inactivation 14,15,37,85,86 . In regards to phenotypic differences observed between sexes and in mice with different genetic backgrounds, it is now known that the gene expression programs of males and females, or that of different mouse strains, are not identical 18,87,88 . Alternative methods for inhibiting gene expression in wild‐type organisms are now available.…”

“… 14 , 15 , 37 , 85 , 86 In regards to phenotypic differences observed between sexes and in mice with different genetic backgrounds, it is now known that the gene expression programs of males and females, or that of different mouse strains, are not identical. 18 , 87 , 88 Alternative methods for inhibiting gene expression in wild‐type organisms are now available. For instance, the inability of cardiomyocytes to utilize fatty acids as a metabolic fuel due to the absence of Fabp3 was previously postulated to be the explanation for exercise intolerance manifested by mice lacking Fabp3.…”