Top-Down Proteomics of Zebrafish Brain Regions Using Capillary Zone Electrophoresis-Tandem Mass Spectrometry

Understanding region-specific proteoform profiles in the brain is crucial for deciphering neural function and identifying therapeutic targets. Zebrafish (Danio rerio) is a valuable vertebrate model for neuroscience research due to its substantial conservation of brain structure and function with that of mammals. We present a label-free quantitative top-down proteomics (TDP) analysis of distinct zebrafish brain regions using microdissection and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS). We analyzed four anatomically distinct regions─telencephalon (Tele), combined habenula-optic tectum (Tec/Hab), cerebellum (Cer), and medulla (Med)─identifying 1,050 proteoforms from 336 proteins. Only 89 proteoforms (5.1%) were shared across all regions, demonstrating substantial proteoform heterogeneity. Quantitative comparisons of proteoform intensity between any two brain regions revealed drastic proteoform abundance differences. Interestingly, proteoforms of the same genes (i.e., sncb, calm1a, mbpa, pcp4l1, apoa2, and nefma) showed opposite expression patterns between brain regions, indicating potential proteoform-specific functions. Nearly 153 neuropeptides were identified using a recently published neuropeptide prediction algorithm with a prediction probability of over 75%, and some neuropeptide proteoforms showed brain-region-specific expression (i.e., pyya, scg2a, and syn1). Gene Ontology analysis of the differentially expressed proteoforms between regions revealed region-specific biological process enrichment, i.e., innate immune response and chromatin organization in Cer, actin organization in Med, microtubule-based processes in Tele, and axonogenesis in Tec/Hab. Comparing quantitative TDP and bottom-up proteomics data from the four zebrafish brain regions revealed substantial discrepancies between proteoform-specific and protein-group-specific data sets, highlighting the value of spatially resolved TDP of brains for better understanding of protein function in a proteoform-specific manner.