Proteomic methodological recommendations for studies involving human plasma, platelets, and peripheral blood mononuclear cells

Baukje De Roos, Susan Joyce Duthie, Abigael C. J. Polley, Francis Mulholland, Freek G. Bouwman, Carolin Heim, Garry Jonathan Rucklidge, Ian T. Johnson, Edwin C. Mariman, Hannelore Daniel, Ruan M. Elliott

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    Abstract

    This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% (p < 0.005) higher recovery of protein in flow-through sample compared with acetone precipitation. The use of OptiPrep gave the lowest level of platelet contamination (1:0.8) during the isolation of PBMC from blood. Several proteins (among which are (x-tropomyosin, fibrinogen and coagulation factor XIII A) were identified that may be used as biomarkers of platelet contamination in future studies. When identifying preselected spots, at least three out of the four centers found similar identities for 10 out of the 10 plasma proteins, 8 out of the 10 platelet proteins and 8 out of the 10 PBMC proteins. The discrepancy in spot identifications has been described before and may be explained by the mis-selection of spots due to laboratory-to-laboratory variation in gel formats, low scores on the peptide analysis leading to no or only tentative identifications, or incomplete resolution of different proteins in what appears as a single abundant spot. The average with in-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual matching improved the CV with on average between 1 and 16%. The average between laboratory CV for each of the matched spots after automatic matching ranged between 4 and 54% for depleted plasma proteins, between 5 and 60% for platelet proteins, and between 18 and 70% for PBMC proteins. This variation must be considered when designing sufficiently powered studies that use proteomics tools for biomarker discovery. The use of tricine in the running buffer for the second dimension appears to enhance the resolution of proteins especially in the high molecular weight range.

    Original languageEnglish
    Pages (from-to)2280-2290
    Number of pages11
    JournalJournal of Proteome Research
    Volume7
    Issue number6
    Early online date20 May 2008
    DOIs
    Publication statusPublished - Jun 2008

    Keywords

    • plasma proteomics
    • platelet proteomics
    • PBMC proteomics
    • human nutrition intervention studies
    • technical variability
    • human endothelial-cells
    • laser-desorption/ionization-time
    • flight mass-spectrometry
    • colon-cancer cells
    • biomarker discovery
    • isolated isoflavones
    • protein expression
    • oxidized-LDL
    • soy extract
    • disease

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