Submitted to: Journal of Chromatography
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/13/2005
Publication Date: 2/2/2006
Citation: Linke, T.N., Ross, C., Harrison, E.H. 2006. Proteomic analysis of rat plasma by two-dimensional liquid chromatography and maldi-tof mass spectrometry. Journal of Chromatography. 1123:160-169. Interpretive Summary: The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers therefore requires either the specific depletion of high abundance proteins using immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe a 2-dimensional (2D) liquid chromatography separation method for the fractionation of rat plasma. In the 1st dimension proteins were separated by chromatofocusing according to their isoelectric point (pI). In the 2nd dimension, proteins were further fractionated by non-porous, reversed phase chromatography according to their hydrophobicity. The data from both separations was displayed as a 2D protein expression map of pI versus retention time (relative hydrophobicity). Both separations were carried out on the ProteomeLab PF 2D system (Beckman Coulter), aninstrument platform that provides a high degree of automation and real-time monitoring of the separation process. We report on the reproducibility of the 2D separation protocol with rat plasma as sample. The peptide and protein composition of each collected fractions was analyzed with MALDI-TOF MS. The performance of this approach in protein expression profiling experiments was tested with plasma samples from vitamin A-sufficient and vitamin A-deficient rats. Vitamin A metabolites (retinoids) are needed for important physiological processes such as vision, reproduction growth, development and immune function. Recent animal studies have have shown that vitamin A deficiency not only affects the expression of genes directly involved in retinoid metabolism such as lecithin:retinol acyltransferase (LRAT) and RA-inducible cytochrome P450 (P450RAI) but also the expression of several liver genes involved in fatty acid metabolism, peroxisomal function, glycolysis, glutamate metabolism and the urea cycle. It is important to point out that retinol regulates the secretion of its own transport protein, retinol binding protein (RBP), from liver into plasma on a post-translational level. Our goal was to detect not only known indicators of vitamin A deficiency such as RBP using the 2D-LC approach but also to identify potentially new biomarkers of vitamin A deficiency by differential protein expression profiling.
Technical Abstract: Here we describe the application of the ProteomeLab PF 2D system from Beckman Coulter in plasma protein expression profiling experiments. The reproducibility of the 1st-dimension separation was evaluated in terms of pH gradient formation. The 2nd-dimension separation was evaluated in terms of peak retention times on the reversed phase column. We found in 4 consecutive chromatofocusing separations that the pH gradient differed by less than 0.2 pH units at any time during the elution step. Second dimension retention times of peaks from identical pI fractions differed by less than 7 seconds in 6 consecutive separations. Quantitation of peptide and protein peaks in terms of peak height and peak area with the integration features of the 32 Karat chromatography software revealed coefficients of variation of less than 10% for high abundance proteins and between 10-20% for medium and low abundance proteins. Each 2D separation generated a total of 540 fractions which were analyzed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) .We detected approximately 275 peptides and proteins with molecular masses ranging from 3 kDa to 225 kDa. Most fractions were found to contain multiple low and high molecular weight proteins. Differential display of 2D protein expression maps from retinol-sufficient and retinol-deficient rat plasma samples identified a fraction with several proteins that appeared to be down-regulated in the vitamin A-deficient animal. This fraction was analyzed by surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) to identify the differentially expressed protein in the sample. We identified a protein with a molecular weight of 44.3 kDa that appeared to be down-regulated in plasma of vitamin A-deficient rats. Quantitative proteomic analysis of complex samples such as plasma is still a difficult task. We discuss the potential of this approach for biomarker discovery and address the experimental challenges that remain.