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

Author(s): Yi J, Liu Z, Craft D, O'Mullan P, Ju G, Gelfand CA

Publication: J Proteome Res, 2008, Vol. 7, Page 5112-8

PubMed ID: 19367699 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of anticoagulant type and protease inhibitors on the stability of FPA and other peptide biomarkers in spiked plasma and serum specimens during room temperature storage. Serum and plasma were prepared within 30-90 min of collection and frozen at -80 degrees C until use. Isotopically labeled peptides were spiked into thawed serum and plasma specimens, which were then incubated at room temperature. Aliquots were taken at specified times for MALDI-TOF mass spectrometric analysis. A subset of blood was collected into BD P100 tubes containing EDTA and proprietary protein stabilizers. Both individual specimens and pooled specimens were used.

   Summary of Findings:

   Isotopically labeled FPA levels declined in all specimens during room temperature storage, but the rates of decline were anticoagulant- and protease inhibitor-dependent. The peak intensity of the FPA peptide was nondetectable after 4 h in heparin plasma, 8 h in citrate plasma, 24 h in EDTA, but was still detectable after 48 h in P100 tubes that contained a protease inhibitor cocktail  FPA was least stable in serum. The authors show that the labeled FPA degradation followed a first-order reaction of kinetics, regardless of protease inhibitors or anticoagulants. Enzymatic degradation occurred almost immediately, as FPA truncated peptides were detectable in heparin plasma at the time zero timepoint. The authors also note that C3f and C4 peptides showed the same relative order of stability among the tested anticoagulants and protease inhibitors. 

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Serum
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Peptide	MALDI-TOF MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	Citrate EDTA Heparin None
   Analyte Extraction and Purification	Protease inhibitor	Cocktail No protease inhibitor added
   Storage	Time at room temperature	0 h 0.5 h 1 h 2 h 4 h 8 h 12 h 24 h 48 h
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
   MALDI-TOF MS Specific	Targeted peptide/protein	FPA C3f C4

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