Proteomics

Dataset Information

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Optimising sample preparation of the plasma peptidome


ABSTRACT: We further expanded on previous optimizations to isolate the plasma peptidome and compared various methods to maximize identifications with speed and ease. Previous studies have reported loss of polypeptides binding to high abundant proteins during depletion strategies. We hypothesized that rapid chaotropic denaturation of plasma with urea under reducing conditions would liberate non-covalently bound peptides to improve recovery during protein depletion. We also compared depletion strategies to isolate the peptidome including protein precipitation and removal with either TCA, acetone or acetonitrile (AcN). Following centrifugation of precipitated proteins, the supernatant containing peptides was collected. For acetone and AcN precipitations, an additional vacuum centrifugation step was required followed by resuspension of peptides in aqueous buffer. Our comparison of peptidome isolation also included removal of proteins with size-exclusion 10 kDa MWCO filters. All peptide isolations were acidified to 0.1% TFA, adjusted to 5% acetonitrile and desalted with HLB-SPE. Peptides were analysed by single-shot nanoUHPLC-MS/MS employing both HCD and EThcD and quantified by LFQ

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Benjamin Parker  

LAB HEAD: Benjamin Parker

PROVIDER: PXD007191 | Pride | 2017-10-09

REPOSITORIES: Pride

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Publications

Multiplexed Temporal Quantification of the Exercise-regulated Plasma Peptidome.

Parker Benjamin L BL   Burchfield James G JG   Clayton Daniel D   Geddes Thomas A TA   Payne Richard J RJ   Kiens Bente B   Wojtaszewski Jørgen F P JFP   Richter Erik A EA   James David E DE  

Molecular & cellular proteomics : MCP 20171005 12


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  ...[more]

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