NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

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

The purpose of this paper was to determine the effects of anticoagulant type and protease inhibitors on the stability of fibrinogen peptide A (FPA) and other peptide biomarkers in spiked plasma and serum specimens during room temperature storage.

Conclusion of Paper

Isotopically labeled FPA levels declined in all specimens during room temperature storage, but the rates of decline were anticoagulant- and protease inhibitor-dependent. FPA was most stable in plasma from BD P100 tubes (with protease inhibitors), followed by EDTA plasma and citrate and heparin plasma (similar rates of degradation). 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. The authors also note that C3f and C4 peptides showed the same relative order of stability among the tested anticoagulants and protease inhibitors.

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
    Preservative Types
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    Peptide MALDI-TOF MS
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(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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