Elsevier

Analytical Biochemistry

Volume 478, 1 June 2015, Pages 14-22
Analytical Biochemistry

Evaluating the effects of preanalytical variables on the stability of the human plasma proteome

https://doi.org/10.1016/j.ab.2015.03.003Get rights and content

Abstract

High quality clinical biospecimens are vital for biomarker discovery, verification, and validation. Variations in blood processing and handling can affect protein abundances and assay reliability. Using an untargeted LC-MS approach, we systematically measured the impact of preanalytical variables on the plasma proteome. Time prior to processing was the only variable that affected the plasma protein levels. LC-MS quantification showed that preprocessing times <6 h had minimal effects on the immunodepleted plasma proteome, but by 4 days significant changes were apparent. Elevated levels of many proteins were observed, suggesting that in addition to proteolytic degradation during the preanalytical phase, changes in protein structure are also important considerations for protocols using antibody depletion. As to processing variables, a comparison of single- vs double-spun plasma showed minimal differences. After processing, the impact ⩽3 freeze–thaw cycles was negligible regardless of whether freshly collected samples were processed in short succession or the cycles occurred during 14–17 years of frozen storage (−80 °C). Thus, clinical workflows that necessitate modest delays in blood processing times or employ different centrifugation steps can yield valuable samples for biomarker discovery and verification studies.

Section snippets

Biospecimen Research Network specimens

Donors gave consent for blood collection for research purposes as part of a UCSF Institutional Review Board-approved protocol. Male and female donors (aged 20–40, median 27, Supplemental Table 1) were requested to fast for a minimum of 12 h prior to blood collection. Donors were seated at least 5 min before the draw and the arm was positioned on a slanting armrest in a straight line from the shoulder to the wrist. A tourniquet was applied approximately 2 inches above the antecubital fossa or

Results

We evaluated the effects of several preanalytical variables related to handling, processing, and storage on the stability of plasma and serum proteomes. To this end, we examined the following variables: time and temperature from specimen collection to processing, plasma centrifugation method, number of freeze–thaw cycles, and time in storage at −80 °C. Effort was made to closely control, during blood collection, all parameters known to impinge on specimens’ properties, i.e., variations in the

Discussion

We conducted a systematic analysis to determine the relationships among blood processing and storage variables and the quality and reproducibility of quantitative proteomics data. Multiple processing and storage variables were assayed: (1) time and temperature before processing; (2) time after thawing prior to immunodepletion; (3) centrifugation steps (single vs double); (4) number of freeze–thaw cycles; and (5) time in frozen storage. An untargeted comparative proteomics approach applied to

Acknowledgments

This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E (UCSF) and PPG Grants P01HD30367, UL1RR024153, and UL1TR000005 (University of Pittsburgh Clinical and Translational Science Institute). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or

References (47)

  • F. Betsou et al.

    Identification of evidence-based biospecimen quality-control tools: a report of the International Society for Biological and Environmental Repositories (ISBER) Biospecimen Science Working Group

    J. Mol. Diagn.

    (2013)
  • A. Hakimi et al.

    Assessment of reproducibility in depletion and enrichment workflows for plasma proteomics using label-free quantitative data-independent LC-MS

    Proteomics

    (2014)
  • H.J. Issaq et al.

    Cancer biomarker discovery: opportunities and pitfalls in analytical methods

    Electrophoresis

    (2011)
  • S.J. Skates et al.

    Statistical design for biospecimen cohort size in proteomics-based biomarker discovery and verification studies

    J. Proteome Res.

    (2013)
  • H.M. Moore et al.

    Biospecimen reporting for improved study quality

    Biopreserv. Biobank.

    (2011)
  • A.J. Rai et al.

    HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples

    Proteomics

    (2005)
  • S.D. Jewell et al.

    Analysis of the molecular quality of human tissues: an experience from the Cooperative Human Tissue Network

    Am. J. Clin. Pathol.

    (2002)
  • M.K. Tuck et al.

    Standard operating procedures for serum and plasma collection: early detection research network consensus statement standard operating procedure integration working group

    J. Proteome Res.

    (2009)
  • J. Yi et al.

    Inhibition of intrinsic proteolytic activities moderates preanalytical variability and instability of human plasma

    J. Proteome Res.

    (2007)
  • L.J. Zimmerman et al.

    Global stability of plasma proteomes for mass spectrometry-based analyses

    Mol. Cell. Proteomics

    (2012)
  • S.A. Randall et al.

    Evaluation of blood collection tubes using selected reaction monitoring MS: implications for proteomic biomarker studies

    Proteomics

    (2010)
  • A. Aguilar-Mahecha et al.

    The effect of pre-analytical variability on the measurement of MRM-MS-based mid- to high-abundance plasma protein biomarkers and a panel of cytokines

    PLoS One

    (2012)
  • J. Marshall et al.

    Processing of serum proteins underlies the mass spectral fingerprinting of myocardial infarction

    J. Proteome Res.

    (2003)
  • Cited by (42)

    • Proteins in the pathway from high red blood cell width distribution to all-cause mortality

      2022, eBioMedicine
      Citation Excerpt :

      In accordance with the guidelines for protein biomarker work, all samples were stored at 4 °C, centrifuged for 4 h, immediately aliquoted, and frozen at −80 °C.7 Plasma proteins remain stable for 14–17 years in storage at −80 °C and for up to 25 freeze–thaw cycles.8,9 The present study measured plasma proteomics at baseline and the 9-year follow-up using the 1·3k HTS SOMAscan assay (SomaLogic, Boulder, CO).10,11

    • Longitudinal Analysis of Circulating Markers of Bone Turnover Across Multiple Decades in Osteoporotic Women

      2022, Journal of Hand Surgery
      Citation Excerpt :

      Each biomarker exhibits different degrees of stability both in vivo and in vitro, and analysis of frozen serum creates the potential for some sample degradation over time. Nonetheless, as reported by Hassis et al,16 human plasma proteins are stable for multiple decades in frozen storage, and the main risk to sample integrity is prolonged time to processing and repeated freeze–thaw cycles. The samples in this study were procured, processed, and archived under stringent Department of Defense protocols and not subject to repeated freeze–thaw cycles.

    View all citing articles on Scopus
    View full text