Global Stability of Plasma Proteomes for Mass

Peptide-based mass spectrometry approaches, such as multiple reaction monitoring, provide a powerful means to measure candidate protein biomarkers in plasma. A poten-tial confounding problem is the effect of preanalytical vari-ables, which may affect the integrity of proteins and pep-tides. Although some blood proteins undergo rapid physiological proteolysis ex vivo, the stability of most plasma proteins to preanalytical variables remains largely unexplored. We applied liquid chromatography-tandem mass spectrometry shotgun proteomics and multiple reac-tion monitoring analyses to characterize the stability of pro-teins at the peptide level in plasma. We systematically eval-uated the effects of delay in plasma preparation at different temperatures, multiple freeze-thaw cycles and erythocyte hemolysis on peptide and protein inventories in prospec-tively collected human plasma. Time course studies indi-cated few significant changes in peptide and protein iden-tifications, semitryptic peptides and methionine-oxidized peptides in plasma from blood collected in EDTA plasma tubes and stored for up to a week at 4 °C or room temper-ature prior to plasma isolation. Similarly, few significant changes were observed in similar analyses of plasma sub-jected to up to 25 freeze-thaw cycles. Hemolyzed samples produced no significant differences beyond the presence of hemoglobin proteins. Finally, paired comparisons of plasma and serum samples prepared from the same patients also yielded few significant differences, except for the depletion of fibrinogen in serum. Blood proteins thus are broadly stable to preanalytical variables when analyzed at the pep-tide level. Collection protocols to generate plasma for mul-tiple reaction monitoring-based analyses may have differ-ent requirements than for other analyses directed at intac