Inter-Regional Proteomic Profiling of the Human Brain Using an Optimized Protein Extraction Method from Formalin-Fixed Tissue to Identify Signaling Pathways

Abstract

Proteomics offers vast potential for studying the molecular regulation of the human brain. Formalin fixation is a common method for preserving human tissue; however, it presents challenges for proteomic analysis. In this study, we compared the efficiency of two different protein-extraction buffers on three post-mortem, formalin-fixed human brains. Equal amounts of extracted proteins were subjected to in-gel tryptic digestion and LC-MS/MS. Protein, peptide sequence, and peptide group identifications; protein abundance; and gene ontology pathways were analyzed. Protein extraction was superior using lysis buffer containing tris(hydroxymethyl)aminomethane hydrochloride, sodium dodecyl sulfate, sodium deoxycholate, and Triton X-100 (TrisHCl, SDS, SDC, Triton X-100), which was then used for inter-regional analysis. Pre-frontal, motor, temporal, and occipital cortex tissues were analyzed by label free quantification (LFQ) proteomics, Ingenuity Pathway Analysis and PANTHERdb. Inter-regional analysis revealed differential enrichment of proteins. We found similarly activated cellular signaling pathways in different brain regions, suggesting commonalities in the molecular regulation of neuroanatomically-linked brain functions. Overall, we developed an optimized, robust, and efficient method for protein extraction from formalin-fixed human brain tissue for in-depth LFQ proteomics. We also demonstrate herein that this method is suitable for rapid and routine analysis to uncover molecular signaling pathways in the human brain.

SSSMuG: Same Sample Sequential Multi-Glycomics

“…A tissue sample preparation that would account for their differing molecular characteristics and solubilities therefore needed to be selected. For mouse brain tissues, there are a wide range of lysis buffers previously used by others, with varying detergents, chaotropes, and salt concentrations. − We chose a simple detergent-free buffer, 300 mM sucrose, and 10 mM Tris–EDTA (STE), with mechanical lysis using a hand-held homogenizer to process the total brain tissue. This lysis and homogenization method also allow preservation of subcellular components such as mitochondria and synaptosomes .…”

Data mining antibody sequences for database searching in bottom-up proteomics

“…Database search was conducted using FragPipe [29][30][31][32][33][34][35][36][37] with the above databases and six PRIDE datasets including four blood plasma datasets (PXD031813, PXD029181, PXD023175, and PXD020354) [38][39][40], two depleted blood plasma datasets (PXD036491 [41] and PXD022296) and one brain dataset (PXD039808) [42]. Depleted blood plasma samples were samples of which most abundant proteins (e.g., antibody, fibrinogen, etc.)…”