Introduction: Mass spectrometry (MS)-based proteomic profiling of human postmortem brain tissue from healthy and disease cases have defined global molecular signatures associated with neurological disease. However, many of these studies primarily focus on the gray matter and discard the oligodendrocyte-rich white matter prior to homogenization and downstream proteomic processing. In this study, we use quantitative proteomics to analyze white and gray matter from the same post-mortem tissue, which highlight unique cell type differences and functional pathways
Methods: Post-mortem prefrontal cortex samples were carefully dissected to separate gray and white matter. Approximately, 50mg of each tissue subtype was homogenized in buffered 8M Urea. An aliquot of 100ug of total protein lysate was subjected to sequential endoproteinase LysC and trypsin digestion. The samples were then labeled with individual tandem mass tags (TMT), combined, and fractionated into 20 fractions using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) offline fractionation. All fractions were run on an Orbitrap Fusion Lumos mass spectrometer using the synchronous precursor selection (SPS)-based MS3 method. All spectra were processed through Proteome Discoverer (version 2.1) and quantitation information was extracted for further differential expression and pathway analysis.
Conclusions
1.Provides a comprehensive quantitative comparison of protein expression between gray and white matter highlighting different pathways of disease relevance.
2.Identification of differentially expressed proteins in both white and gray matter from patients with Alzheimer’s disease.
3.Identification of various pathways with distinct relationship to white matter.