A method for the extraction of the endogenous tryptic peptides (peptidome) from human EDTA plasma

Analytical Biochemistry
Jaimie DufresneJohn Marshall

Abstract

The proteins identified from endogenous peptides agreed between serum versus plasma, and tryptic versus non-tryptic peptides, when collected by C18 alone and analyzed by liquid chromatography electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) including amyloids, apolipoproteins, haptoglobin, complements, fibrinogens, hemopexin, antitrypsin and alpha 2 macroglobulin. Precipitation of polypeptides from plasma in 9 vol of 100% organic solvent followed by stepwise extraction of the insoluble pellet with an increasing fraction of water identified thousands of proteins. A Coomassie-blue protein binding assay, and tricine SDS-PAGE, showed that Acetonitrile-Water (AH) resulted in a greater relative enrichment of low molecular weight plasma polypeptides than Acetonitrile-Methanol Water (AMH). A total of 905,386 MS/MS spectra greater than ~10,000 (E4) counts were correlated by X!TANDEM to a federated human protein library of 153,124 different protein sequences that resulted in 58,223 fully tryptic peptides from 3463 Gene Symbols of which 1880 had ≥ 5 independent peptides (p ≤ 0.00001). The results were filtered and organized in an SQL database for analysis using the generic R statistical analysis system. Cellular protein...Continue Reading

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Citations

Jan 2, 2019·Clinical Proteomics·Jaimie DufresneJohn G Marshall
Jan 1, 2020·Clinical Proteomics·Jaimie DufresneJohn G Marshall
Jul 9, 2020·Clinical Proteomics·Thanusi ThavarajahJohn G Marshall
Dec 7, 2018·Clinical Proteomics·Jaimie DufresneJohn G Marshall
Jun 30, 2021·Clinical Proteomics·Angelique Florentinus-MefailoskiJohn G Marshall

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