A Tiered Approach to Human Synapse Proteomics: Optimized LC-MS/MS Analysis of Whole-Tissue and Synaptosome Preparations from Frozen Post-mortem Brain Samples

Recent advancements in neuroproteomics have enabled detailed analysis of protein expression and function in the human brain. Post-mortem human brain studies have significantly advanced our understanding of the relationship between genetics, cell biology of neurological and psychiatric disorders and their clinical diagnosis. Synaptic dysfunction often has a central role in these disorders. Therefore, we specifically evaluated the sensitivity of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect synaptic proteins in whole-tissue lysates versus synaptosome preparations. First, we optimized sample preparation protocols for frozen human gray matter (GM), refining the suspension TRAPping (sTRAP) digestion method to improve protein solubilization and Cysteine reduction and alkylation using thin human tissue sections, and to accomplish low technical variation by minimizing sample handling. We achieved a highly reproducible sample preparation workflow by rigorously applying standardization and randomization across dissection, processing, and LC-MS/MS runs. Second, comparative LC-MS/MS analysis showed that cortical whole-tissue lysates are a practical solution for large-scale studies and broadly detected synaptic proteins focusing on excitatory neurons. However, enrichment by synaptosome isolation offered improved resolution of synapse-specific proteins. Because synapse-proteomics enables insight into spatial regulation--i.e., alterations at the synapse that are not reflected in the soma-we recommend a tiered approach: initial whole-tissue analysis for broad disease-associated changes, followed by targeted synaptosome proteomics to deepen insight into synaptic alterations. This strategy optimally balances throughput, reproducibility, and biological relevance, and enhances the study of brain disorders through proteomics. Moreover, analyzing synaptic proteins first at the tissue level improves insight into overall regulation of synaptic proteins induced by synapse loss or gain.