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

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.

ANGT_HUMAN[448–462], an Anorexigenic Peptide Identified Using Plasma Peptidomics

“…As a result, 129 synthetic peptides were judged appropriate for biological validation experiments. Many of these peptides were used in our previous screening study to test their biological activities in vascular cells and in vivo experiments, which led to the discovery of 3 suprabasin-derived peptides (SBSN_HUMAN[225-237], SBSN_HUMAN[243-259], and SBSN_HUMAN[279-295]) [ 5 ] and a proGIP-derived peptide (GIP_HUMAN[22-51]) [ 20 ]. The biological activities of the remaining 125 peptides were screened using a variety of mammalian cells in culture, and experiments were performed to examine their ability to induce the expression of genes downstream of major intracellular signaling molecules, to increase [Ca 2+ ] i , and to induce mitogenesis.…”

“…The obtained low-molecular-weight peptide fractions extracted from 800 μL of human plasma were lyophilized and redissolved in LC-MS/MS-compatible surfactants and separated by reverse-phase (RP)-HPLC, collected at 1-minute intervals and combined into 8 fractions by the cyclic sample pooling method [ 5 , 19 ]. The extracts derived from 80 μL of plasma were analyzed using a quadrupole Orbitrap benchtop mass spectrometer, Q-Exactive (Thermo Fisher Scientific), equipped with an EASY-nLC 1000 system (Thermo Fisher Scientific) essentially as described previously [ 20 ]. The peptides were injected directly onto an analytical column (C18, particle diameter 3 μm, 75 μm i.d.…”

“…The dynamic exclusion time was set at 30 seconds. Plasma concentrations were extrapolated from the extracted ion chromatograms (XICs) generated using the respective endogenous peptides and the corresponding spiked, stable isotope-labeled peptides [ 20-22 ].…”

LC–MS peak assignment based on unanimous selection by six machine learning algorithms

“…Shotgun proteomics using high-resolution MS enables us to conduct MS1-based quantitative comparisons of objective and control peptides from the XIC 1 , 2 . To identify the proteins involved in physiologic and/or pathologic processes based on abundance using shotgun proteomics, poor chromatographic peaks must be excluded from complex LC–MS/MS spectra when using conventional criteria, such as idotP and ∆M 8 – 12 , and then quantifications based on the areas of extracted peaks of identified proteins can be compared.…”

“…Liquid chromatography–mass spectrometry (LC–MS) has advanced remarkably in recent years, and LC–MS-based shotgun proteomics techniques enable the comprehensive identification and quantification of tryptic peptides. Further developments in high-resolution MS capabilities have enabled MS1-based quantitative comparisons of objective and control peptides from extracted ion chromatograms (XICs) 1 , 2 . Shotgun proteomics techniques are thus commonly used in biological research (e.g., identification of disease-specific biomarkers) 3 – 5 .…”