Quantitative and comparative mass spectrometric analysis of high-frequency versus low-frequency areas in the auditory brainstem by means of SILAM
A basic organization principle of the auditory system is the tonotopy, by which high- and low-frequent acoustic signals (hf versus lf) are perceived in anatomically distinct areas. The tonotopic organization is present in virtually all nuclei of the auditory brainstem. We hypothesize that specific sets of proteins reflect the special requirements for hf versus lf signal processing, for example kinetics of receptors and action potentials. Disclosing these sets of proteins is essential for comprehending how acoustic signal processing (and possibly neuronal signal processing in general) is achieved. Thus far, molecular differences between hf and lf areas have been addressed for single candidate proteins only (e.g. Kv3.1b channels), but not in a systemic way. Kv1.3 channels are more abundant in the hf areas and result in short action potential duration, a prerequisite of hf information processing. Here, we aim to identify and quantify the set of proteins in the hf and lf areas of four auditory brainstem regions of rats or mice: the dorsal cochlear nucleus, the medial nucleus of the trapezoid body, the lateral superior olive, and the inferior colliculus. To do so, we will employ laser-assisted microdissection (in collaboration with Prof. Thomas Deller, Frankfurt) to accurately collect the anatomically small areas of interest. Identification and relative quantification of proteins will be performed using SWATH mass spectrometry (in collaboration with Prof. Michael Schroda) and stable isotope labeling of amino acids in mammals (SILAM). SILAM brains are commercially available, and we will pursue a ratiometric approach, i.e., we will determine the ratio of ratio. One great advantage of SILAM is that there is no discrimination against membrane or low level proteins. The set of identified proteins will be screened for candidates that are exclusively expressed or significantly more abundant in one frequency area. Functional annotation and further statistical analyses will result in biological functions or pathways that are overrepresented in either the hf or the lf area. Differences regarding single protein candidates will be validated via independent methods, such as Western blots or immunohistochemistry. Differences regarding functional terms shall be addressed by electrophysiological approaches. We hope that our analyses will provide fundamentally new aspects into the understanding of hf and lf processing in the auditory brainstem and in the brain in general.