Two-dimensional liquid chromatography/tandem mass spectrometry analysis of Gradiflow fractionated native human plasma

Valerie C. Wasinger (Lead / Corresponding author), Vicki L. Locke, Mark J. Raftery, Mark Larance, Deborah Rothemund, Audrey Liew, Irene Bate, Michael Guilhaus

    Research output: Contribution to journalArticlepeer-review

    23 Citations (Scopus)

    Abstract

    One of the major challenges facing protein analysis is the dynamic range of protein expression within massively complex samples (Corthals, G. L. et al.., Electrophoresis 2000, 21, 1104-1115). In plasma this difference is as great as ten orders of magnitude, and this is currently beyond the range of detection achievable by any of the analytical techniques. Plasma has the additional challenge of having a few highly abundant proteins, such as albumin, which mask the detection of lower abundance and biologically significant proteins. The use of the Gradiflow BF400 as a fractionation tool to deplete highly abundant albumin from human plasma is reported here. A sequential three-step protocol was performed on five plasma samples as part of the International Plasma Proteome Project organised by the HUPO; four containing different anticoagulants: EDTA, citrate, heparin and a control sample (NIBSC); and a serum sample. Plasma from an alternate source also underwent fractionation and served as an in-house control. Time modulation between 1 and 7 h was observed for the depletion of albumin from these samples. Following albumin depletion, each fraction was trypsin-digested and the peptides were fractionated further using a 2-D LC-MS/MS. Differences in the total number of proteins identified for each sample were also noted.
    Original languageEnglish
    Pages (from-to)3397-3401
    Number of pages5
    JournalProteomics
    Volume5
    Issue number13
    DOIs
    Publication statusPublished - Aug 2005

    Keywords

    • Mass Spectrometry
    • Blood Proteins
    • Anticoagulants
    • Trypsin
    • Chromatography
    • Electrophoresis, Polyacrylamide Gel
    • Albumins
    • Humans
    • Citric Acid
    • Immunoprecipitation
    • Proteomics
    • Proteome
    • Chromatography, Liquid
    • Peptides
    • Time Factors
    • Heparin
    • Edetic Acid

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