Stable isotope metabolic labeling with a novel N-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 (15)N bacteria diet based on Ralstonia eutropha protein, which was applied to a mouse model for trait anxiety. Tissue from (15)N-labeled vs. (14)N-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 (15)N 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 (15)N bacteria diet provides researchers a novel tool to discover disease-relevant protein expression differences in mouse models using quantitative mass spectrometry.

Figure 1. Mouse feeding protocol with the novel bacteria diet.

As soon as pregnancy was detected 10 days after mating (post natal day PND -11), the animals received 4 days of ad libitum standard/bacteria diet before they were fed bacteria diet only. Organs were harvested at PND 5, 14, 28 and 56 to determine ¹⁵N incorporation rate and line-specific protein expression pattern.

Figure 2. Body weight after weaning of animals fed with bacteria, blue-green algae and standard diet.

Compared to standard fed HAB animals (orange dotted line), blue-green algae diet fed animals were significantly heavier (green line) and bacteria fed animals lighter (red line) (**p<0.01 blue-green algae vs. control ++p<0.01 bacteria vs. control).

Figure 3. Incorporation rate of ¹⁵N in (A) plasma and (B) brain proteins.

In 5, 14 and 28 day old animals (PND 5, 14, 28) bacteria diet feeding provides a significantly higher incorporation of ¹⁵N in plasma proteins compared to blue-green algae diet; only at the age of 56 days, blue-green algae fed diet animals show the same ¹⁵N incorporation. In the cerebellum, a faster incorporation in adolescence at PND 14 reached significance (**p<0.01; *p<0.05).

Figure 4. Anxiety-related behavior and locomotion of HAB animals on the elevated plus maze (EPM).

No behavioral changes due to the blue-green algae diet fed to the animals were found.

Figure 5. Depression-like behavior of HAB animals in the tail suspension test (TST).

Animals fed with blue-green algae diet hat a strongly reduced immobility, indicating depression-like behavior, compared to standard fed animals. This effect was even more pronounced in animals fed with the ¹⁵N isotope (*p<0.05; **p<0.01).

Figure 6. (A) Anxiety-related behavior at post natal day 5 in the ultrasonic vocalization test (USV).

High (HAB), normal (NAB) and low (LAB) anxious animals were fed with ¹⁴N or ¹⁵N enriched bacteria diets. A) Independent of the diet, HAB, NAB and LAB mice showed the significant (not indicated) phenotypic divergence typical of animals of the standard breeding (dotted lines and indicated in B) as percentage difference of the standard breeding).

Figure 7. Anxiety-related behavior and locomotion on the elevated plus maze (EPM).

High (HAB), normal (NAB) and low (LAB) anxious animals were fed with ¹⁴N or ¹⁵N enriched bacteria diets. A) HAB, NAB and LAB mice showed the significant (not indicated) phenotypic divergence typical of animals of the standard breeding (dotted lines and indicated in B) as percentage difference of the standard breeding).

Figure 8. Depression-like behavior in the tail suspension test (TST).

High (HAB), normal (NAB) and low (LAB) anxious animals were fed with ¹⁴N and/or ¹⁵N enriched bacteria diets. A) No behavioral changes due to diet per se were found for animals fed with bacteria diet compared to the animals of the respective lines of the HAB/NAB/LAB standard breeding of the same generation (dotted lines and indicated in B) as percentage difference of the standard breeding). However, ¹⁵N fed HAB animals showed a strongly reduced immobility, indicating depression-like behavior, compared to ¹⁴N fed HABs (**p<0.01). B) This differed significantly from the standard HAB/NAB/LAB breeding (*p<0.05). Due to division by zero, no value is given for ¹⁴N fed LAB animals in relation to the standard breeding: values are ¹⁴N LAB 0.53±0.3 sec vs. standard LAB 4.06±1.5 sec.

Figure 9. Analysis of expression differences by MS analysis.

A) ESI mass spectrum of the ¹⁴N LAB (red) and ¹⁵N HAB (blue) isotope forms of the GLO1 peptide GFGHIGIAVPDVYSACK in cerebellar tissue. The isotopologue patterns of the ¹⁴N and ¹⁵N peptide signals (m/z) were used for relative quantification with ProRata. B) Extracted ion chromatograms for the GLO1 peptide, extracted from A, showing an upregulation of GLO1 in LAB mice. No smoothing was applied.

Figure 10. Analysis of expression differences by MS analysis and RT-PCR.

A) ESI mass spectrum of the ¹⁴N LAB (red) and ¹⁵N HAB (blue) isotope forms of the Hint2 peptide ISQAEEDDQQLLGHLLLVAK in cerebellar tissue. The isotopologue patterns of the ¹⁴N and ¹⁵N peptide signals (m/z) were used for relative quantification with ProRata. B) Extracted ion chromatograms for the Hint2 peptide, extracted from A, showing an upregulation of Hint2 in HAB mice. C) Analysis of Hint2 transcripts in ¹⁵N-labeled HAB and ¹⁴N LAB cerebellar cDNA by RT-PCR, normalized by HRSP12 expression. Hint2 expression in HABs was 2.5-fold higher compared to LABs (*p<0.05). No smoothing was applied.