Stable Isotope Metabolic Labeling with a Novel 15N-Enriched Bacteria Diet for Improved Proteomic Analyses of Mouse Models for Psychopathologies

Abstract

The identification of differentially regulated proteins in animal models of psychiatric diseases is essential for a comprehensive analysis of associated psychopathological processes. Mass spectrometry is the most relevant method for analyzing differences in protein expression of tissue and body fluid proteomes. However, standardization of sample handling and sample-to-sample variability are problematic. Stable isotope metabolic labeling of a proteome represents the gold standard for quantitative mass spectrometry analysis. The simultaneous processing of a mixture of labeled and unlabeled samples allows a sensitive and accurate comparative analysis between the respective proteomes. Here, we describe a cost-effective feeding protocol based on a newly developed 15N bacteria diet based on Ralstonia eutropha protein, which was applied to a mouse model for trait anxiety. Tissue from 15N-labeled vs. 14N-unlabeled mice was examined by mass spectrometry and differences in the expression of glyoxalase-1 (GLO1) and histidine triad nucleotide binding protein 2 (Hint2) proteins were correlated with the animals' psychopathological behaviors for methodological validation and proof of concept, respectively. Additionally, phenotyping unraveled an antidepressant-like effect of the incorporation of the stable isotope 15N into the proteome of highly anxious mice. This novel phenomenon is of considerable relevance to the metabolic labeling method and could provide an opportunity for the discovery of candidate proteins involved in depression-like behavior. The newly developed 15N bacteria diet provides researchers a novel tool to discover disease-relevant protein expression differences in mouse models using quantitative mass spectrometry.

Quantitative assessment of the impact of the gut microbiota on lysine ε-acetylation of host proteins using gnotobiotic mice

“…The field of quantitative epiproteomics in gnotobiotic animals is currently in its infancy but holds great promise for enhancing our understanding of how various microbial communities impact the biology of their hosts. (13,42,43) was generated by mixing spirulina lyophilisate (Cambridge Isotope Laboratories) with proteinfree diet (Harlan Teklad). Additional information regarding the composition of these diets can be found in Tables S1 and S2.…”

Protein Turnover Quantification in a Multilabeling Approach: From Data Calculation to Evaluation

“…With our method, we can, moreover, address a severe problem of metabolic labeling approaches: often it is impossible to reach a high rate, let alone a complete, incorporation of stable isotopes, which is in particular true for higher organisms such as Eukaryotes (71). Although initially developed for pulse-chase experiments, our algorithm is also capable of successfully quantifying proteins having an uncertain and diversified incorporation rate of isotopic labels such as heavy stable nitrogen or carbon.…”

O potencial da rotulação metabólica de 15N para a pesquisa de esquizofrenia

“…rodents) to achieve high 15 N incorporation rates for quantitative proteomics comparisons. Importantly, the introduction of 15 N may have an effect on the behavioral phenotype 4 or on protein expression levels 10 . As a consequence, labeling controls (either using internal labeled standards or reciprocal labeling) should be implemented in the experimental design to avoid artifacts caused by the 15 N isotope affecting relative protein quantification results.…”

“…In the context of psychiatry, a 15 N metabolic labeling protocol via a bacteria-based diet was established and applied to study the mouse model of high (HAB), normal (NAB) and low (LAB) anxiety-related behavior 4 . 15 N-labeled NAB mice were used as internal standards and quantitative proteomics studies in cingulate cortex, hippocampus and plasma were performed to compare HAB and LAB mice.…”