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 C-terminal peptides from Transgelins, which were increased in plasma during exercise. In vitro 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.
Highlights
From the ‡Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, NSW 2006, Australia; §School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia; ¶Department of Nutrition, Exercise and Sports, August Krogh Centre, University of Copenhagen, Copenhagen 2100, Denmark; ʈSchool of Medicine, University of Sydney, Sydney, NSW 2006, Australia
To identify differentially regulated peptides between two or more states, quantitative peptidomic analysis has been performed using label-free approaches (7), stable isotope labeling with chemical derivatisation (8, 9), or stable isotope labeling with metabolic incorporation (10)
The TCA precipitation method was the fastest, simplest and cheapest procedure directly compatible with hydrophilic-lipophilic balance solid-phase extraction (HLB-SPE)
Summary
From the ‡Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, NSW 2006, Australia; §School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia; ¶Department of Nutrition, Exercise and Sports, August Krogh Centre, University of Copenhagen, Copenhagen 2100, Denmark; ʈSchool of Medicine, University of Sydney, Sydney, NSW 2006, Australia. One of the most common methods of communication involves the secretion or release of proteins and peptides from one cell in response to an environmental perturbation These signals travel via the blood to modulate physiological pathways in other cells and tissues. A variety of methods have been used to isolate the peptidome including molecular weight separation techniques such as size-exclusion chromatography or filtration (1, 2) or depletion of larger proteins with acid precipitation or organic solvents (3, 4) The combination of these extraction techniques with multidimensional liquid chromatography has led to the identification of hundreds of endogenous low molecular weight peptides in plasma (5). We hypothesize that characterizing the plasma peptidome in response to exercise will allow an investigation of protease regulation and simultaneously identify new signaling factors that contribute to physiological adaptions Such an analysis will provide another step forward in validating the use of peptidomics as a useful tool for discovery of both novel regulatory factors and clinical diagnostics. The unexplored complexity of the exercise peptidome may reveal important signaling molecules mediating the beneficial effects of exercise
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