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We investigated the effect of homogenization strategy and protein precipitation on downstream protein quantitation using multiple reaction monitoring mass spectrometry (MRM-MS). Our objective was to develop a workflow capable of processing disparate tissue types with high throughput, minimal variability, and maximum purity. Similar abundances of endogenous proteins were measured in nine different mouse tissues regardless of the homogenization method used; however, protein precipitation had strong positive effects on several targets. The best throughput was achieved by lyophilizing tissues to dryness, followed by homogenization via bead-beating without sample buffer. Finally, the effect of tissue perfusion prior to dissection and collection was explored in 20 mouse tissues. MRM-MS showed decreased abundances of blood-related proteins in perfused tissues; however, complete removal was not achieved. Concentrations of nonblood proteins were largely unchanged, although significantly higher variances were observed for proteins from the perfused lung, indicating that perfusion may not be suitable for this organ. We present a simple yet effective tissue processing workflow consisting of harvest of fresh nonperfused tissue, novel lyophilization and homogenization by bead-beating, and protein precipitation. This workflow can be applied to a range of mouse tissues with the advantages of simplicity, minimal manual manipulation of samples, use of commonly available equipment, and high sample quality.

Monitoring the reproductive characteristics of a species can complement existing conservation strategies by understanding the mechanisms underlying demography. However, methodology to determine important aspects of female reproductive biology is often absent in monitoring programs for large mammals. Protein biomarkers may be a useful tool to detect physiological changes that are indicative of reproductive state. This study aimed to identify protein biomarkers of reproductive status in serum collected from free-ranging female brown bears (Ursus arctos) in Alberta, Canada, from 2001 to 2018. We hypothesized that the expression of proteins related to reproduction in addition to energetics and stress can be used to answer specific management-focused questions: (i) identify when a female is pregnant, (ii) detect if a female is lactating, (iii) determine age of sexual maturity (i.e. primiparity) and (iv) assess female fertility (i.e. reproduction rate). Furthermore, we investigated if silver spoon effects (favourable early life conditions provide fitness benefits through adulthood) could be determined using protein expression. A target panel of 19 proteins with established relationships to physiological function was measured by peptide-based analysis using liquid chromatography and multiple reaction monitoring mass spectrometry and their differential expression was evaluated using a Wilcoxon signed-rank test. We found biomarkers of pregnancy (apolipoprotein B-100 and afamin), lactation (apolipoprotein B-100 and alpha-2-macroglobulin) and sexual maturity (corticosteroid-binding globulin), but there were no statistically significant relationships with protein expression and fertility. The expression of proteins related to reproduction (afamin) and energetics (vitamin-D binding protein) was associated with the nutritional quality of the individual’s present habitat rather than their early life habitat. This study highlights potential biomarkers of reproductive status and provides additional methods for monitoring physiological function in wildlife to inform conservation.

MotivationLaboratory mouse is the most used animal model in biological research, largely due to its high conserved synteny with human. Researchers use mice to answer various questions ranging from determining a pathological effect of knocked out/in gene to understanding drug metabolism. Our group developed >5000 quantitative targeted proteomics assays for 20 mouse tissues and determined the concentration ranges of a total of >1600 proteins using heavy labeled internal standards. We describe here MouseQuaPro; a knowledgebase that hosts this collection of carefully curated experimental data. ResultsThe web-based application includes protein concentrations from >700 mouse tissue samples from three common research strains, corresponding to >200k experimentally determined concentrations. The knowledgebase integrates the assay and protein concentration information with their human orthologs, functional and molecular annotations, biological pathways, related human diseases and known gene expressions. At its core are the protein concentration ranges, which provide insights into (dis)similarities between tissues, strains and sexes. MouseQuaPro implements advanced search as well as filtering functionalities with a simple interface and interactive visualization. This information-rich resource provides an initial map of protein absolute concentration in mouse tissues and allows guided design of proteomics phenotyping experiments. The knowledgebase is available on mousequapro.proteincentre.com. Availability and implementation The knowledgebase is available free of charge on http://mousequapro.proteincentre.com. Supplementary information Supplementary data are available at Bioinformatics online.

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.

The plasma compartment of the blood holds important information on the risk to develop cardiovascular diseases such as venous thrombosis (VT). Mass spectrometry-based targeted proteomics with internal standards quantifies proteins in multiplex allowing generation of signatures associated with a disease or a condition. Here, to demonstrate the method, we investigate the plasma protein signatures in mice following the onset of VT, which was induced by RNA interference targeting the natural anticoagulants antithrombin and protein C. We then study mice lacking Slc44a2, which was recently characterized as a VT-susceptibility gene in human genome-wide association studies. We use a recently developed panel of 375 multiplexed mouse protein assays measured by mass spectrometry. A strong plasma protein siganture was observed when VT was induced. Discriminators included acute phase response proteins, and proteins related to erythrocyte function. In mice lacking Slc44a2, protein signature was primarily overruled by the difference between sexes and not by the absent gene. Upon separate analyses for males and females, we were able to establish a signature for Slc44a2 deficiency, in which glycosylation-dependent cell adhesion molecule-1 and thrombospondin-1 were shared by both sexes. The minimal impact of Slc44a2 deficiency on the measured plasma proteins suggests that the main effect of Slc44a2 on VT does not lay ultimately in the plasma compartment. This suggests further investigation into the role of this VT-susceptibility gene should perhaps also question the possible involvement in cellular mechanisms.

Large carnivores play critical roles in the maintenance and function of natural ecosystems; however, the populations of many of these species are in decline across the globe. Therefore, there is an urgent need to develop novel techniques that can be used as sensitive conservation tools to detect new threats to the health of individual animals well in advance of population-level effects. Our study aimed to determine the expression of proteins related to energetics, reproduction and stress in the skin of grizzly bears (Ursus arctos) using a liquid chromatography and multiple reaction monitoring mass spectrometry assay. We hypothesized that a suite of target proteins could be measured using this technique and that the expression of these proteins would be associated with biological (sex, age, sample location on body) and environmental (geographic area, season, sample year) variables. Small skin biopsies were collected from free-ranging grizzly bears in Alberta, Canada, from 2013 to 2019 (n = 136 samples from 111 individuals). Over 700 proteins were detected in the skin of grizzly bears, 19 of which were chosen as targets because of their established roles in physiological function. Generalized linear mixed model analysis was used for each target protein. Results indicate that sample year influenced the majority of proteins, suggesting that physiological changes may be driven in part by responses to changes in the environment. Season influenced the expression of proteins related to energetics, reproduction and stress, all of which were lower during fall compared to early spring. The expression of proteins related to energetics and stress varied by geographic area, while the majority of proteins that were affected by biological attributes (age class, sex and age class by sex interaction) were related to reproduction and stress. This study provides a novel method by which scientists and managers can further assess and monitor physiological function in wildlife.

When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.

Targeted proteomics using multiple reaction monitoring (MRM) has become the method of choice for the rapid, precise, and reproducible quantitation of proteins in complex biological matrices. MRM-based targeted proteomics allows protein profiling in complex biological samples and accurate comparisons of between samples [1-6], typically using a bottom-up LC-MS/MS approach [7]. While relative quantitation approaches report differences in fold-changes between the samples being compared, absolute methods measure the concentrations [8], ideally based on isotopically labeled internal standard peptides that are analogues of the endogenous proteotypic target peptides [9]. The use of stable-isotope labeled standard (SIS) peptides is a key factor in the precision and reproducibility of absolute quantitation experiments using LC/MRM-MS, but the results may still not be accurate. Experimentally derived concentrations for specific tissues or biofluids can vary as a function of digestion protocol, or even which peptides are selected to represent a given protein [10-12]. This article is protected by copyright. All rights reserved