Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7556911 | Analytical Biochemistry | 2018 | 34 Pages |
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 9â¯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 905,386â¯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 hadâ¯â¥â¯5 independent peptides (pâ¯â¤â¯0.00001). The results were filtered and organized in an SQL database for analysis using the generic R statistical analysis system. Cellular proteins including secreted and exosome proteins, signaling factors, nucleic acid binding proteins, metabolic enzymes and uncharacterized factors were observed with a significant enrichment of expected protein-protein interactions by STRING analysis.
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Authors
Jaimie Dufresne, Angelique Florentinus-Mefailoski, Pete Bowden, John G. Marshall,