An inherent problem with the analysis of ‘bulk’ tissue by traditional proteomics methods is that the cellular heterogeneity of a tissue is often unaddressed. This heterogeneity results in averaging of the signals generated by the constituent cells and the data are dominated by those cells that are the most abundant. Isolating cells of a single type by laser capture microdissection alleviates the signal averaging caused by measuring a mixed population of cell types. Here we developed a simple, quantitative sample preparation method for sub-1000 cells isolated by laser capture microdissection to isolate Purkinje cells from histological tissue slices of cerebellum obtained from post-mortem human donors.
We were able to reproducibly detect over 2000 proteins from only 200 Purkinje cells isolated from histological tissue slices. The overlap in protein identifications was 80 % between two replicates from different tissue slices within the same donor. The proteomes generated were deep enough to detect proteins involved in Purkinje cell development and function along with almost 600 proteins involved in general neurological cell processes. The method also shows good quantitative reproducibility between replicates (Pearson Correlation Coefficient = 0.90).
Biography of Roman Fischer
Dr. Fischer leads the Discovery Proteomics Facility and is a principal investigator at the University of Oxford, UK. His lab focusses on proteomic strategies to access the deep proteome from minimal sample materials, such as cells harvested by laser-capture-microdissection. Employing a series of recently published techniques (GASP, CHOPIN) they aim at not only comprehensive proteome-, but also proteome sequence coverage to detect protein isoforms and PTMs in the context of tumor biology and drug target discovery.