Isolating and identifying native peptides in serum samples can be like finding a needle in a haystack, or as a recent paper in Scientific Reports described it “selecting and identifying several hundred sesame seeds randomly scattered in an Olympic-size swimming pool filled with an enormous variety of beans”. This is because these low molecular weight peptides are vastly underrepresented among high abundance serum proteins, like albumin and immunoglobulins (“the beans”), and are not always represented in traditional tryptic protein databases, which complicates analysis of LC-MS/MS data from these peptides. Taguchi and colleagues addressed the challenge by refining a previous published differential solubilization method for peptide extraction combined with sample prefractionation and a cyclic sample pooling technique. This methodology combined with PEAKS de novo-assisted database + PTM search resulted in the identification of almost 8000 distinct native peptides. Using information from this data set, the researchers selected a subset of approximately 100 high quality peptides to test for biological activity using synthetic polypeptides and a variety of cell-based assays. These screening assays identified a subset of bioactive peptides originating from the suprabasin protein. Additional experiments confirmed binding of the suprabasin-derived peptides to the surface of cells, the peptides’ tissue distribution, effect on behaviour in mice, and nM peptide concentrations in human plasma. This well-rounded study showed that the research group’s approach to plasma peptidomics can identify unknown low abundance peptides with biological activity, including three novel peptides originating from the same precursor protein.
How was PEAKS used?
PEAKS Studio X was used for de novo-assisted database searching of LC-MS/MS data. The inclusion of de novo sequencing directly in the PEAKS workflow means that sequence information and a quality score is available for native peptides not found in the chosen database. Integrated PEAKS PTM search was used to identify variable modifications and substitutions.
Taguchi T, Kodera Y, Oba K, Saito T, Nakagawa Y, Kawashima Y, Shichiri M. Suprabasin-derived bioactive peptides identified by plasma peptidomics. Sci Rep. 2021 Jan 13;11(1):1047. doi:10.1038/s41598-020-79353-4. PMID: 33441610; PMCID: PMC7806982.
Identification of low-abundance, low-molecular-weight native peptides using non-tryptic plasma has long remained an unmet challenge, leaving potential bioactive/biomarker peptides undiscovered. We have succeeded in efficiently removing high-abundance plasma proteins to enrich and comprehensively identify low-molecular-weight native peptides using mass spectrometry. Native peptide sequences were chemically synthesized and subsequent functional analyses resulted in the discovery of three novel bioactive polypeptides derived from an epidermal differentiation marker protein, suprabasin. SBSN_HUMAN[279–295] potently suppressed food/water intake and induced locomotor activity when injected intraperitoneally, while SBSN_HUMAN[225–237] and SBSN_HUMAN[243–259] stimulated the expression of proinflammatory cytokines via activation of NF-κB signaling in vascular cells. SBSN_HUMAN[225–237] and SBSN_HUMAN[279–295] immunoreactivities were present in almost all human organs analyzed, while immunoreactive SBSN_HUMAN[243–259] was abundant in the liver and pancreas. Human macrophages expressed the three suprabasin-derived peptides. This study illustrates a new approach for discovering unknown bioactive peptides in plasma via the generation of peptide libraries using a novel peptidomic strategy.