Blood collection protocols are a crucial pre-analytical variable that must be carefully defined during the development of any reproducible biomarker assay. It is essential to limit endogenous proteolysis during collection and preservation of the plasma sample in a state compatible with down-stream analysis. This study tested the proteome and peptidome stability of matched plasma samples collected from healthy individuals. Each set of samples was donated simultaneously and stored/handled identically using three different types of blood collection tubes; (i) spray-dried EDTA (ii) EDTA with polymer gel, and (iii) EDTA tubes containing a proprietary cocktail of protease inhibitors (P100TM). All samples were investigated with proteomic and peptidomic approaches using 2D difference gel electrophoresis (2D DIGE) and solid phase extraction (SPE) followed by ESI-MS/MS (Electrospray Ionization) analysis. Results from 2D DIGE showed no significant influence of the different anticoagulant tubes on the level of plasma proteins observed by 2D DIGE, although minor variations in one protein per individual in each individual were observed, which statistically appears to be a random occurrence. In contrast, results obtained from the peptidomic analysis demonstrated a lower number of endogenous peptides suggesting preservation of some proteins in plasma treated with the protease inhibitor cocktail. However every plasma sample analysed showed endogenous peptides obtained from six proteins (namely Fibrinogen alpha chain, Complement C4-A, Complement C4-B, Alpha-2-antiplasmin, Complement C3 and Apolipoprotein E) irrespective of the anticoagulant used for collection. These data should assist researchers in their choice of ideal anticoagulants for plasma proteome and peptidome research.
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Mahboob, S., Ahn, S. B., Mohamedali, A., Amirkhani, A., Tan, S., Ranganathan, S., ... Baker, M. S. (2016). Addition of protease inhibitors to EDTA containing blood collection tubes does not deliver significant advantage for preservation of plasma for down-stream analysis. Clinical Proteomics and Bioinformatics, 1(3), 1-9. https://doi.org/10.15761/CPB.1000115