Multiplexed Temporal Quantification of the Exercise-regulated Plasma Peptidome

Abstract

Exercise is extremely beneficial to whole body health reducing the risk of a number of chronic human diseases. Some of these physiological benefits appear to be mediated via the secretion of peptide/protein hormones into the blood stream. The plasma peptidome contains the entire complement of low molecular weight endogenous peptides derived from secretion, protease activity and PTMs, and is a rich source of hormones. In the current study we have quantified the effects of intense exercise on the plasma peptidome to identify novel exercise regulated secretory factors in humans. We developed an optimized 2D-LC-MS/MS method and used multiple fragmentation methods including HCD and EThcD to analyze endogenous peptides. This resulted in quantification of 5,548 unique peptides during a time course of exercise and recovery. The plasma peptidome underwent dynamic and large changes during exercise on a time-scale of minutes with many rapidly reversible following exercise cessation. Among acutely regulated peptides, many were known hormones including insulin, glucagon, ghrelin, bradykinin, cholecystokinin and secretogranins validating the method. Prediction of bioactive peptides regulated with exercise identified -terminal peptides from Transgelins, which were increased in plasma during exercise. experiments using synthetic peptides identified a role for transgelin peptides on the regulation of cell-cycle, extracellular matrix remodeling and cell migration. We investigated the effects of exercise on the regulation of PTMs and proteolytic processing by building a site-specific network of protease/substrate activity. Collectively, our deep peptidomic analysis of plasma revealed that exercise rapidly modulates the circulation of hundreds of bioactive peptides through a network of proteases and PTMs. These findings illustrate that peptidomics is an ideal method for quantifying changes in circulating factors on a global scale in response to physiological perturbations such as exercise. This will likely be a key method for pinpointing exercise regulated factors that generate health benefits.

Peptidomic analysis of endogenous plasma peptides from patients with pancreatic neuroendocrine tumours

“…This protein is believed to be involved in the secretory process of regulatory peptides and secreted peptides from VGF were identified in islets . These peptides have previously been reported in plasma extracts from an exercise study . None of the peptides that were identified have been classed as bioactive peptides from the VGF pro‐protein …”

“…A study by Parker et al used 200 μL of plasma from multiple time-points that were combined using a tandem mass tag protocol. 10 Intact peptides from the proglucagon gene were detected in this study but only the glicentin-related polypeptide (GRPP) was identified after significant sample workup. In some clinical settings it is not feasible or practical to obtain large plasma sample volumes, and analysis methodologies would be required to do more with less starting material.…”

“…Whilst this method proved to be effective, it did not detect the peptides intact (protease digestion was performed), therefore complicating data interpretation. A study by Parker et al used 200 μL of plasma from multiple time‐points that were combined using a tandem mass tag protocol . Intact peptides from the proglucagon gene were detected in this study but only the glicentin‐related polypeptide (GRPP) was identified after significant sample workup.…”

“…Although these previous methodologies have shown effectiveness at extracting peptides from plasma, they are not considered high throughput, and in the case of immunoaffinity extraction, have to be performed consecutively. A high‐throughput approach for enriching the low molecular weight (LMW) plasma peptidome/proteome is the use of protein precipitation – either through solvent‐ or acid‐based precipitation . The organic solvent approach has demonstrated the capability of detecting peptides in plasma in the low pg/mL range on its own or with a subsequent SPE phase using selected reaction monitoring (SRM)‐based analyses .…”

“…A high-throughput approach for enriching the low molecular weight (LMW) plasma peptidome/proteome is the use of protein precipitationeither through solvent-or acid-based precipitation. 9,10 The organic solvent approach has demonstrated the capability of detecting peptides in plasma in the low pg/mL range on its own 11 or with a subsequent SPE phase using selected reaction monitoring (SRM)-based analyses. 12,13 These approaches have shown that plasma pre-treatment combined with highly targeted triple quadrupole analysis is capable of detecting specific peptides at low pg/mL concentrations, although very few untargeted peptidomics analyses have been published.…”