15N metabolic labeling of high and low anxiety-related behavior HAB/LAB mice

To elucidate the pathophysiology of anxiety disorders, we try to identify proteins underlying increased trait anxiety. Protein expression levels of high (HAB), normal (NAB) and low (LAB) anxiety-related behavior mice are studied using quantitative mass-spectrometry. Comparing two groups of animals, we labeled all proteins of one group with 15N to analyze 15N/14N-signal pairs, allowing a highly sensitive comparison of protein expression. For 15N-labeling, we fed HAB mice from fetal status on with a special 15N-enriched Spirulina diet. This resulted in 15N-incorporation of around 90% in all proteins of the organism. The special Spirulina nutrition had no effect on the inbred trait anxiety of the mice, comparing 15N-, 14N-, and standard diet fed animals in the elevated plus maze test. To identify expression differences in the brain, protein preparations of subcellular compartments and subproteomes of interest were fractionated and analyzed by LC-MALDI TOF/TOF or LC-ESI-Orbitrap mass spectrometry. Subsequently, the relative quantitation was done by summing up the isotope peak intensities for the 14N- and the 15N-protein and comparing the signal pairs using our QuantiSpec software. Next we will compare protein expression levels of naïve vs. stressed HAB, NAB, and LAB mice with and without pharmacological treatment. The identified proteins have the potential to provide new targets for preclinical research and clinical treatment of anxiety disorders.