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.