Fig. 1 |. Schematic depicting characterization of peripheral blood mononuclear cell proteoforms from whole blood by top-down proteomics.
The protocol described here has been optimized for the performance of translational top-down proteomics from a single ~8-mL draw of human blood. Whole blood is subsequently fractionated into its component parts by density-gradient centrifugation, and the PBMCs, which are highly relevant to immune-related pathologies, are isolated and prepared for analysis. The key value proposition of top-down proteomics is that it analyzes proteoforms, rather than peptides yielded by tryptic or other enzymatic digestion. This allows the analyst to characterize the specific post-translational modifications and other chemical and genetic sequence variants in a sample of whole proteome that would be lost by bottom-up proteomics, but that may have high mechanistic relevance to the phenotypes of interest. As depicted in the figure inset marked by the dashed box, a single gene product often gives rise to multiple proteoforms after post-translational modification or because of the existence of sequence variants derived from proteolytic processing, multiple isoforms, or even single-nucleotide polymorphisms.