The glucose-dependent insulinotropic polypeptide (GIP) is secreted from enteroendocrine cells and have 42-amino-acid. It can enhance the release of insulin from pancreatic cells. The identification and amino acid sequencing of these peptides in human plasma is a challenging task and is barely studied by the scientists because of the difficulties associated with analysis of peptides in plasma samples. Identifying plasma bioactive peptides and disease biomarkers remains tricky not only because of the trace levels of these compounds, but also the fact that the plasma contains an extraordinarily large dynamic range of high-molecular-weight proteins.

In this study a novel technology is established that can improve the plasma extraction yield, leading to the identification of trace peptides with low molecular weight using a single drop of plasma sample. Also, to find the peptide that can modulate the cardiovascular system, the identification results were synthesised, and their bioactivities were investigated. As a result, a 30-amino-acid proatherogenic peptide, GIP_HUMAN [22–51] was introduced as a potent proatherosclerotic peptide hormone.

How was PEAKS used?

Initially, the PEAKS Studio (version 7) was used to perform peak picking, de-isotoping, charge deconvolution of fragment ions and a de novo peptide sequencing-based database search from the MS and MS/MS spectra of peptides. In this study, 485 built-in modifications were explored. These analyses were conducted against the SwissProt_2015_02.fasta database until the beginning of 2018. They resulted in the identification of 18,552 polypeptide sequences with a peptide filter of 1% for false discovery rate (FDR). The large number of PTMs limited the accuracy of PTM search and the Mascot MS/MS ion search was used which can show high scores for peptides longer than 30 amino acids despite having poor MS/MS spectra. To solve these issues a secondary search was conducted using PEAKS Studio (version X).

In the second trial, the data were reanalysed and a database search was performed against the SwissProt_2020_03.fasta (selected for Homo sapiens; 20,365 entries). Here again PEAKS studio performed peak picking, de-isotoping, charge deconvolution of fragment ions and a de novo peptide sequencing-based database search from the MS and MS/MS spectra of peptides. The search parameters were: enzyme, no enzyme; fixed modification, carbamidomethyl (C, only for samples with reductive alkylation); variable modifications, acetyl (N-term, K), amidated (C-term), pyro-glu from Q (Q), oxidation (M), carbamidomethyl (N-terminal, only for samples with reductive alkylation); peptide ion mass tolerance was set at 6 ppm with fragment ion mass tolerance of 0.02 Da and 0 or 1% FDR. Tis process resulted in the identification of 7,959 or 11,256 distinct native peptide sequences depending on FDRs of 0% or 1%, respectively.

Masaki, Tsuguto, et al. “GIP_HUMAN [22–51] is a new proatherogenic peptide identified by native plasma peptidomics.” Scientific Reports 11.1 (2021): 1-14. https://doi.org/10.1038/s41598-021-93862-w

Abstract

We recently established a new plasma peptidomic technique and comprehensively identified a large number of low-molecular weight and low-abundance native peptides using a single drop of human plasma. To discover a novel polypeptide that potently modulates the cardiovascular system, we performed a bioinformatics analysis of the large-scale identification results, sequentially synthesized the selected peptide sequences, tested their biological activities, and identified a 30-amino-acid proatherogenic peptide, GIP_HUMAN[22–51], as a potent proatherosclerotic peptide hormone. GIP_HUMAN[22–51] has a common precursor with the glucose-dependent insulinotropic polypeptide (GIP) and is located immediately N-terminal to GIP. Chronic infusion of GIP_HUMAN[22–51] into ApoE^−/− mice accelerated the development of aortic atherosclerotic lesions, which were inhibited by co-infusions with an anti-GIP_HUMAN[22–51] antibody. GIP_HUMAN[22–51] increased the serum concentrations of many inflammatory and proatherogenic proteins, whereas neutralising antibodies reduced their levels. GIP_HUMAN[22–51] induced IκB-α degradation and nuclear translocation of NF-κB in human vascular endothelial cells and macrophages. Immunoreactive GIP_HUMAN[22–51] was detected in human tissues but there was no colocalization with the GIP. The plasma GIP_HUMAN[22–51] concentration in healthy humans determined using a stable-isotope tagged peptide was approximately 0.6 nM. This study discovered a novel endogenous proatherogenic peptide by using a human plasma native peptidomic resource.