We have previously reported that intrinsic protease activity in plasma and serum samples cause ex vivo variability and instability of samples (Yi et al, J. Proteome Res. (2007) 6: 1768-1781). In this study, we investigated stability of peptide biomarkers in human plasma and serum. Biomarkers were tested in five blood samples: serum or plasma with either citrate, heparin, or EDTA as anticoagulant, or EDTA plus protease inhibitors (BD P100*). After incubation of the sample spiked with a peptide (FPA, GLP-1, BNP, C3 and C4) for a specified period ranging from 0 minute to 72 hours, the peptides were extracted and analyzed by MALDI-TOF MS. The results indicate that all tested biomarkers are decreasing over time, demonstrating their proteolytic instability in these different blood products. Within the same serum or plasma sample, each peptide shows a unique half life, suggesting that the peptidase specificity differs between peptides. However, the spiked peptides are the most stable in the protease-inhibited plasma by inhibition of intrinsic proteolysis. The stability of the peptides in different collected samples is as following order: P100 plasma > EDTA > Citrate & Heparin > Serum. Furthermore, kinetic analysis of the fragments of digested FPA suggests that the intrinsic peptidase activity cause a first-order, sequential multiple step reaction (SMSR). The modeling analysis of the SMSR indicates a significant contribution of the end leaving reside on the stability of the peptide. The applications of these observations to plasma proteome research will be further discussed.

Footnote: * For research use only.