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Proteomic approaches to vitamin A research. Protein expression analysis of retinol‐deficient rat plasma by two‐dimensional liquid chromatography
Author(s) -
Linke Thomas,
Ross A. Catharine,
Harrison Earl H.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a997-a
Subject(s) - chromatofocusing , chromatography , chemistry , isoelectric point , isoelectric focusing , retinol binding protein , mass spectrometry , fractionation , fetuin , blood proteins , protein precipitation , retinol , high performance liquid chromatography , affinity chromatography , vitamin , biochemistry , glycoprotein , enzyme
Proteomic analysis of plasma or serum is a formidable challenge due to the presence of a few highly abundant proteins. Detection of low abundance protein biomarkers requires either the specific depletion of high abundance proteins with immunoaffinity columns and/or other protein prefractionation methods. Here we describe a 2‐dimensional liquid chromatography separation method for the fractionation of rat plasma. In the 1 st dimension proteins were separated by chromatofocusing according to their isoelectric point. In the 2 nd dimensions, proteins were fractionated by non‐porous, reversed‐phase chromatography according to hydrophobicity. Data from both separations was displayed as a protein expression map of isoelectric point versus hydrophobicity. In 4 consecutive chromatofocusing separations, the pH gradient differed by less than 0.2 pH units during elution. Second dimension peak retention times differed by less than 7 seconds in 6 consecutive separations. Each sample was separated into a total of 540 fractions which were analyzed by mass spectrometry. We detected about 275 proteins with molecular masses ranging from 3 to 225 kDa. Differential display of 2D protein expression maps from authenticated retinol‐sufficient and retinol‐deficient plasma samples identified a protein that was consistently down‐regulated in vitamin A‐deficient rats. Mass spectrometric analyses revealed differential expression of a 44 kDa protein tentatively identified as β‐fetuin. Supported by NIH and USDA.