Characterization of the low-molecular-weight human plasma peptidome
The human plasma proteome represents an important secreted sub-proteome. Proteomic analysis of blood plasma with mass spectrometry is a challenging task. The high complexity and wide dynamic range of proteins as well as the presence of several proteins at very high concentrations complicate the profiling of the human plasma proteome. The peptidome (or low-molecular-weight fraction, LMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based markers of disease. Peptides are generated by active synthesis and proteolytic processing, often yielding proteolytic fragments that mediate a variety of physiological and pathological functions. As such, degradomic studies, investigating cleavage products via peptidomics and top-down proteomicsÂ in particular, have warranted significant research interest. However, due to their molecular weight, abundance, and solubility, issues with identifying specific cleavage sites and coverage of peptide fragments remain challenging. Peptidomics is currently focused toward comprehensively studying peptides cleaved from precursor proteins by endogenous proteases. This protocol outlines a standardized rapid and reproducible procedure for peptidomic profiling of human plasma using centrifugal ultrafiltration and mass spectrometry. Ultrafiltration is a convective process that uses anisotropic semipermeable membranes to separate macromolecular species on the basis of size. We have optimized centrifugal ultrafiltration (cellulose triacetate membrane) for plasma fractionation with respect to buffer and solvent composition, centrifugal force, duration, and temperature to facilitate recovery >95% and enrichment of the human plasma peptidome. This method serves as a comprehensive and facile process to enrich and identify a key, underrepresented sub-proteome of human blood plasma.
This work was supported, in part, by the National Health and Medical Research Council (NHMRC) of Australia project grant #1057741 (R.J.S.), La Trobe University Leadership RFA Grant (D.W.G. ), and La Trobe Institute for Molecular Science Biomedical Fellowship (D.W.G.).
Book TitleSerum/Plasma Proteomics : Methods and Protocols
EditorsGreening DW Simpson RJ
Place of publicationNew York
SeriesMethods in Molecular Biology
Pagination17p. (p. 63-79)
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Science & TechnologyLife Sciences & BiomedicineBiochemical Research MethodsBiochemistry & Molecular BiologyBloodPlasmaProteomeLow-molecular weightLMFLMWUltrafiltrationPeptidomeProteomicsDegradomeSPECTROMETRY-BASED SERUMMASS-SPECTROMETRYPROTEOME-PROJECTPROTEINSULTRAFILTRATIONSAMPLESELECTROPHORESISIDENTIFICATIONCHROMATOGRAPHYRESOLUTIONHumansPeptidesElectrophoresis, Polyacrylamide GelMolecular WeightTandem Mass SpectrometryStatistics as TopicBiomarkersDevelopmental Biology