The proteins cleaved by endogenous tryptic proteases in normal EDTA plasma by C18 collection of peptides for liquid chromatography micro electrospray ionization and tandem mass spectrometry

J. Dufresne, A. Florentinus-Mefailoski, J. Ajambo, A. Ferwa, P. Bowden and J.  Marshall

Clin Proteomics. 2017; 14: 39. Published online 2017 Dec 2. doi: 10.1186/s12014-017-9174-9


Abstract

The tryptic peptides from ice cold versus room temperature plasma were identified by C18 liquid chromatography and micro electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Samples collected on ice showed low levels of endogenous tryptic peptides compared to the same samples incubated at room temperature. Plasma on ice contained peptides from albumin, complement, and apolipoproteins and others that were observed by the X!TANDEM and SEQUEST algorithms. In contrast to ice cold samples, after incubation at room temperature, greater numbers of tryptic peptides from well characterized plasma proteins, and from cellular proteins were observed. A total of 583,927 precursor ions and MS/MS spectra were correlated to 94,669 best fit peptides that reduced to 22,287 correlations to the best accession within a gene symbol and to 7174 correlations to at least 510 gene symbols with ≥ 5 independent MS/MS correlations (peptide counts) that showed FDR q-values ranging from E−9 (i.e. FDR = 0.000000001) to E−227. A set of 528 gene symbols identified by X!TANDEM and SEQUEST including C4B showed ≥ fivefold variation between ice cold versus room temperature incubation. STRING analysis of the protein gene symbols observed from endogenous peptides in normal plasma revealed an extensive protein-interaction network of cellular factors associated with cell signalling and regulation, the formation of membrane bound organelles, cellular exosomes and exocytosis network proteins. Taken together the results indicated that a pool of cellular proteins, or protein complexes, in plasma are apparently not stable and degrade soon after incubation at room temperature.

Acknowledgements

This research was paid for by the Fonds National de la Recherche, Mobility of Researcher award through Luxembourg Institute of Health LIH (formerly CRP Sante), Luxembourg to JGM and a Grant from the Integrated Biobank of Luxembourg. We gratefully acknowledge the review and critique of this manuscript by Dr. Fay Betsou and Dr. R.A. Phillips of IBBL.

Funding

This research was paid for by the Fonds National de la Recherche, Mobility of Researcher award through Luxembourg Institute of Health LIH (formerly CRP Sante) and the Integrated Biobank of Luxembourg (IBBL) to JGM.

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