Project PXD006501

PRIDE Assigned Tags:
Biological Dataset Biomedical Dataset

Summary

Title

Quantitative proteomic analysis of the human extracellular vesicle proteome reveals a novel mechanism for tissue cross-talk during exercise

Description

Exercise stimulates the release of a plethora of molecules into the circulation, supporting the concept that inter-tissue signaling proteins are important mediators of adaptations to exercise. Recognising that many circulating proteins might be packaged in extracellular vesicles (EV), we employed quantitative proteomic techniques to characterise the exercise-induced secretion of EV contained proteins. We observed an increase in circulation of over 300 proteins, with a notable enrichment of several classes of proteins that compose exosomes and small vesicles, in human arterial blood samples. Pathway analyses revealed significant enrichments in a multitude of biological processes and signalling pathways. Pulse chase and intravital imaging experiments suggest EV-mediated muscle-liver cross talk during exercise. Moreover, by employing arterio-venous balance studies across the contracting human limb, we identified several novel candidate myokines. These data offer a new paradigm by which tissue cross-talk during exercise can exert systemic biological effects

Sample Processing Protocol

For method optimisation, extracellular vesicles were isolated from plasma samples of three healthy human volunteers via 2 x 1h centrifugation at 20,000g or 100,000g in double volume PBS. For the human exercise study, plasma samples were obtained from indwelling femoral arterial and venous catheters in healthy male participants carrying out 1 hour of cycle exercise. Thawed plasma samples were centrifuged x2 at 20,000g for 1h in double volume PBS for isolatation of vesicles. Vesicles were lysed in denaturing buffer and reduced, alkylated and digested in solution by 10mM DTT, 25mM IAA and 1:20 LysC/Trypsin respectively. For the SILAC pulse-chase experiment, Lys6 SILAC mouse exosomes were isolated by ultracentrifugation and AML12 hepatocytes were treated with either the resultant exosome fraction, the vesicle depeleted plasma or PBS. After a 4h incubation, cells were washed and lysed in denaturing buffer. Following acetone precipitation, protein was digested as described above. Peptides were separated into 7 fractions via SAX using STAGE tips packed in house using strong anion exhange disks. All peptide samples were desalted and where relevant fractionated using SDB-RPS stage tips. Peptides were resuspended in loading buffer containing 2% acetonitrile, 0.5% acetic acid and loaded onto a 50cm x 75μm inner diameter column packed in house with 1.9μm C18AQ particles (Dr Maisch GmbH HPLC) using an Easy nLC-1000 UHPLC operated in single column mode loading at 700bar. Peptides were separated using a 180min linear gradient at a flow rate of 200nl/min using buffer A (0.1% formic acid) and a 5-30% buffer B (80% Acetonitrile, 0.1% formic acid). MS data were acquired on a Q Exactive classic operated in data dependent mode. MS spectra were acquired at 70,000 resolution, m/z range of 300-1750 and a target value of 3e6 ions, maximum injection time of 100ms. The top 20 precursor ions were isolated for MS/MS spectra after fragmentation with 2m/z isolation, 8.3e5 intensity threshold, normalised collision energy of 30 at 17,500 resolution at 200m/z, a 60ms injection time and 5e5 AGC target.

Data Processing Protocol

For label free analyses, raw data files were searched against the human Uniprot database, downloaded on 27/09/16, using Maxquant v1.5.5.1. Data were analysed with variable (Oxidation (M), Acetyl (Protein N Term) and fixed (carbamidomethyl (C) modifications with label free quantitation and match between runs functions enabled. Protein quantitation was carried out on a minimum ratio count of 1. Quantitative data were extracted from the protein groups files and analysed in Perseus (v 1.5.5.3). For the SILAC experiments, raw data derived from hepatocyte fractions were searched against the mouse Uniprot database downloaded on 25/7/16 in Maxquant. Data were analysed with variable (Oxidation (M), Acetyl (Protein N Term) and fixed (carbamidomethyl (C) modifications with Lys6 labels and requantify, match between runs functions enabled. Peptide quantitation was carried out on a minimum of 2 peptide ratios. For all data sets, all proteins failing FDR but with a modified site, present in the decoy(reverse) database or known contaminants were filtered out prior to statistical analyses.

Contact

Martin Whitham, Garvan Institute of Medical Research
Martin Whitham, Diabetes & Metabolism, Garvan Institute of Medical Research ( lab head )

Submission Date

28/09/2017

Publication Date

10/01/2018

Cell Type

hepatocyte

Instrument

Q Exactive

Software

Not available

Experiment Type

Shotgun proteomics

Publication

    Whitham M, Parker BL, Friedrichsen M, Hingst JR, Hjorth M, Hughes WE, Egan CL, Cron L, Watt KI, Kuchel RP, Jayasooriah N, Estevez E, Petzold T, Suter CM, Gregorevic P, Kiens B, Richter EA, James DE, Wojtaszewski JFP, Febbraio MA. Extracellular Vesicles Provide a Means for Tissue Crosstalk during Exercise. Cell Metab. 2018 27(1):237-251.e4 PubMed: 29320704