Suprabasin-derived bioactive peptides identified by plasma peptidomics

Abstract

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.

GIP_HUMAN[22–51] is a new proatherogenic peptide identified by native plasma peptidomics

“…As described in the Methods section, we reanalysed these resources using the PEAKS Studio (version X), and a database search was performed against the SwissProt_2020_03.fasta with five variable PTMs in addition to the carbamidomethylation without using the PTM algorithm. This process resulted in the identification of 7,959 or 11,256 distinct native peptide sequences depending on FDRs of 0% or 1%, respectively 22 . We performed a bioinformatic analysis of this updated resource to select bioactive peptide candidates for chemical synthesis and functional validation using the criteria described in the Methods section.…”

“…2 b), but not in the HAoSMCs. Of the remaining 128 peptides, two suprabasin-derived peptides elicited significant cellular responses in the HAoSMCs 22 . Three other peptides induced marginal increases in [Ca 2+ ] i in the HAoECs; however, their detailed biological activity has not subsequently been investigated.…”

“…6a'-l'), and undetected in lung, pituitary, spleen, pancreas, adrenal gland, spine, thyroid, bone marrow, fallopian tube, skin, thymus, uterus, bladder, ovary and prostate. GIP_HUMAN [22] is biosynthesised theoretically, concomitantly with GIP from their common precursor protein, and GIP is expressed abundantly in a limited number of organs, such as the small intestine and duodenum. Therefore, we investigated whether GIP and GIP_HUMAN peptides were co-localised www.nature.com/scientificreports/ within cells by co-staining the two peptides.…”

“…However, to complete the discovery process of putative peptide hormones, their exact native amino acid sequences in the peripheral circulation needs to be confirmed by mass spectrometry 20 . Despite long-term endeavours to elucidate the plasma proteome 11 , 21 , the comprehensive identification of plasma low-molecular weight native peptides has not been achieved until very recently 22 .…”

“…In a recent study, we established improved technology that improved the high-yield plasma extraction technique 22 , enabling the large-scale identification of plasma native peptides with mass spectrometry. We deposited the results into a newly generated human plasma native peptidomic database.…”