Stable isotope metabolic labeling of a mouse model reveals synaptic biomarkers for anxiety disorders

Anxiety disorders affect 20% of the general population. To identify protein biomarkers for anxiety disorders, we have compared the proteomes of high (HAB), normal (NAB) and low (LAB) anxiety-related behavior mouse models by stable isotope metabolic labeling and quantitative mass spectrometry. Mice were metabolically labeled with a 15N-enriched diet. Labeling efficiency was assessed at different developmental time points resulting in up to 92% 15N incorporation in brain tissue. To evaluate isotope-related expression differences synaptosomal proteins of 15N-labeled and unlabeled HAB mice were compared. To detect phenotype-related differences unlabeled HAB and LAB synaptosomal proteins were compared with 15N NAB proteins. To quantify protein expression levels, labeled and unlabeled cortices were mixed and the synaptosomal fraction enriched. After SDS-PAGE fractionation and in-gel tryptic digestion, peptides were analyzed by LC-MS/MS and 14N/15N peptide pairs were used for relative quantification. Isotope- as well as line-specific comparisons showed significant protein variations in cortex synaptosomes between HAB, NAB and LAB mice that are validated by immunochemical analyses. Regulated proteins reveal candidate biomarkers as well as pathways involved in anxiety disorders and contribute to our understanding of anxiety pathophysiology.