Often serving as the initial point of contact between our research project investigators (users) and the Neurometabolomics and Neuroproteomics Center on Cell-Cell Signaling, the Sampling and Separation Core focuses on developing and advancing sample preparation and fractionation techniques for in-depth chemical analysis of brain neurochemistry across various tissue, cell, and subcellular levels. Accurate and precise sampling is essential for successful proteome, peptidome, and metabolome investigations. Unlike DNA/RNA- based molecular techniques, mass spectrometry (MS) analysis of proteins and metabolites in volume- and mass- limited samples lacks a significant signal amplification step. This Core offers unmatched capabilities in the sampling of tissues, cells, and organelles, enabling the chemical characterization of various classes of molecules from an ever-increasing range of samples and animal models. Our philosophy is that directed sampling is fundamental to reducing the intrinsic chemical and morphological complexity of heterogeneous neuronal systems to achieving effective measurement of dynamic brain systems. The methodological advances we have developed already provide better results for experiments that, in some cases, would have been considered impossible to perform without these approaches. A unifying strategy for studies employing separation methods is that they seek to fractionate complex tissue samples into components that make them more accessible for in- depth chemical characterization via MS analysis in the Molecular Profiling and Characterization Core. Since our inception, we have expanded our analytical toolset to include a number of both well-established and innovative approaches for analyte sampling and separation, thus providing robust processing of nervous system samples for metabolite, peptide, and protein characterization. Obviously, this Core is closely linked to the needs of our users, and exists within the framework of the entire Center. Only when integrated with the Molecular Profiling and Characterization Core, and the Bioinformatics, Data Analytics and Predictive Modeling Core, and also the Pilot Research Project Core, are we able to generate the biological information required by our users. Simply stated, the Sampling and Separation Core links the biological user base to the rest of the Center and lays the foundation for successful Center-to-user project outcomes. The close interactions between the neuroscience users and the Sampling Core chemists and biologists enables a robust synergy. Our user base has become increasingly aware of the advantages provided by the analytical tools and expertise available to them through our Core. So too have the facility chemists gained greater familiarity with the diversity of sample types, from a fruit fly brain to a cow brain, with sample sizes ranging from whole brain extracts to single cells, to individual organelles. We integrate the methodologies with the neuroscience to ens...