Proteomic approaches to vitamin A research. Protein expression analysis of retinol‐deficient rat plasma by two‐dimensional liquid chromatography

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.