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J Proteome Res. 2008 Dec;7(12):5112-8. doi: 10.1021/pr800396c.

Intrinsic peptidase activity causes a sequential multi-step reaction (SMSR) in digestion of human plasma peptides.

Author information

1
BD Diagnostics, and BD Medical, One Becton Drive, Franklin Lakes, New Jersey 07417, USA. Jizu_Yi@bd.com

Abstract

Human plasma and serum samples, including protein and peptide biomarkers, are subjected to preanalytical variations and instability caused by intrinsic proteases. In this study, we directly investigated the stability of peptide biomarkers by spiking an isotopically labeled peptide into human plasma and serum samples and then monitoring its time-dependent change. Fibrinogen peptide A (FPA) was used as a model substrate, and its degradation in a conventional serum and plasma either with citrate, heparin, or EDTA as the anticoagulant, or EDTA plus protease inhibitors (inhibited plasma), was measured using time-course MALDI-TOF MS analysis. The FPA and other peptides tested in this study vary in these samples. However, the peptides are most stable in the inhibited plasma followed by, in general order, EDTA plasma, citrate plasma, heparin plasma and serum, demonstrating the benefit of plasma versus serum, and protease inhibitors for biomarker stabilization. Kinetic analysis indicates that intrinsic peptidases cause an observed first-order Sequential Multiple-Step Reaction (SMSR) in digestion of the peptide. Modeling analysis of the SMSR demonstrates that step reactions differ in their kinetic rate constants, suggesting a significant contribution of the truncated end residue on the substrate specificity of the intrinsic peptidase(s). Our observations further show that synthetic peptides introduced into plasma as internal controls can also be degraded, and thus, their (in)stability as a preanalytical variable should not be overlooked.

PMID:
19367699
DOI:
10.1021/pr800396c
[Indexed for MEDLINE]

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