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ABSTRACT: 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 requires therefore either the specific depletion of high abundance proteins with immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe the depletion of seven abundant rat plasma proteins with an immunoaffinity column with coupled antibodies directed against albumin, IgG, transferrin, IgM, haptoglobin, fibrinogen and alpha1-anti-trypsin. The IgY-R7-LC2 (Beckman Coulter) column showed high specificity for the targeted proteins and was able to efficiently remove most of the albumin, IgG and transferrin from rat plasma samples as judged by Western blot analysis. Depleted rat plasma protein samples were analyzed by SELDI-TOF MS, 2D SDS-PAGE and 2D-LC and compared to non-depleted plasma samples as well as to the abundant protein fraction that was eluted from the immunoaffinity column. Analysis of the depleted plasma protein fraction revealed improved signal to noise ratios, regardless of which proteomic method was applied. However, only a small number of new proteins were observed in the depleted protein fraction. Immunoaffinity depletion of abundant plasma proteins results in the significant dilution of the original sample which complicates subsequent analysis. Most proteomic approaches require specialized sample preparation procedures during which significant losses of less abundant proteins and potential biomarkers can occur. Even though abundant protein depletion reduces the dynamic range of the plasma proteome by about 2-3 orders of magnitude, the difference between medium-abundant and low abundant plasma proteins is still in the range of 7-8 orders of magnitude and beyond the dynamic range of current proteomic technologies. Thus, exploring the plasma proteome in greater detail remains a daunting task.
Journal of Chromatography B 05/2007; 849(1-2):273-81. · 2.89 Impact Factor
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ABSTRACT: 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 two-dimensional (2D) liquid chromatography separation method for the fractionation of rat plasma. In the first dimension proteins were separated by chromatofocusing according to their isoelectric point (pI). In the second 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), an instrument platform that provides a high degree of automation and real-time monitoring of the separation process. The reproducibility of the first-dimension separation was evaluated in terms of pH gradient formation. The second-dimension separation was evaluated in terms of peak retention times on the reversed-phase column. We found in four 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 s in six consecutive separations. 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 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 -deficient rat plasma samples identified a fraction with several proteins that appeared to be down-regulated in the vitamin A-deficient animal. 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.
Journal of Chromatography 09/2006; 1123(2):160-9. · 4.53 Impact Factor
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ABSTRACT: Previous work demonstrated both acid and neutral, bile salt-independent retinyl ester hydrolase activities in rat liver homogenates. Here we present the purification, identification, and characterization of an acid retinyl ester hydrolase activity from solubilized rat liver microsomes. Purification to homogeneity was achieved by sequential chromatography using SP-Sepharose cation exchange, phenyl-Sepharose hydrophobic interaction, concanavalin A-Sepharose affinity and Superose 12 gel filtration chromatography. The isolated protein had a monomer molecular mass of approximately 62 kDa, as measured by mass spectrometry. Gel filtration chromatography of the purified protein revealed a native molecular mass of approximately 176 kDa, indicating that the protein exists as a homotrimeric complex in solution. The purified protein was identified as carboxylesterase ES-10 (EC 3.1.1.1) by N-terminal Edman sequencing and extensive LC-MS/MS sequence analysis and cross-reaction with an anti-ES-10 antibody. Glycosylation analysis revealed that only one of two potential N-linked glycosylation sites is occupied by a high mannose-type carbohydrate structure. Using retinyl palmitate in a micellar assay system the enzyme was active over a broad pH range and displayed Michaelis-Menten kinetics with a K(m) of 86 microm. Substrate specificity studies showed that ES-10 is also able to catalyze hydrolysis of triolein. Cholesteryl oleate was not a substrate for ES-10 under these assay conditions. Real time reverse transcriptase-PCR and Western blot analysis revealed that ES-10 is highly expressed in liver and lung. Lower levels of ES-10 mRNA were also found in kidney, testis, and heart. A comparison of mRNA expression levels in liver demonstrated that ES-10, ES-4, and ES-3 were expressed at significantly higher levels than ES-2, an enzyme previously thought to play a major role in retinyl ester metabolism in liver. Taken together these data indicate that carboxylesterase ES-10 plays a major role in the hydrolysis of newly-endocytosed, chylomicron retinyl esters in both neutral and acidic membrane compartments of liver cells.
Journal of Biological Chemistry 07/2005; 280(24):23287-94. · 4.77 Impact Factor
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ABSTRACT: The recent development of high-throughput proteomic technologies has given us new methods to analyze how an organism responds to changes in its nutritional environment. The analysis of plasma samples by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was investigated as a novel approach to the identification of new biomarkers of nutrient status. Pre-fractionation of rat plasma by anion-exchange chromatography in 96-well filter plates markedly increased the total number of unique peptides and proteins that could be observed in SELDI-TOF mass spectra. Replicate fractionations generated nearly identical pH fractions, not only in terms of peptide and protein composition but also in respect to the ion signal intensity of replicate SELDI-TOF mass spectra. The feasibility of this approach was tested with samples from retinol-sufficient and retinol-deficient rats. The comparative analysis revealed reduced levels of three proteins with molecular masses between 10,000 and 20,000 in plasma of retinol-deficient rats. These results demonstrate that plasma profiling by anion-exchange fractionation and SELDI-TOF-MS may be a promising surveillance tool to detect changes in nutritional status and whole body physiology.
Journal of Chromatography 08/2004; 1043(1):65-71. · 4.53 Impact Factor
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ABSTRACT: The recent development of high-throughput proteomic technologies has given us new methods to analyze how an organism responds to changes in its nutritional environment. The analysis of plasma samples by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI–TOF–MS) was investigated as a novel approach to the identification of new biomarkers of nutrient status. Pre-fractionation of rat plasma by anion-exchange chromatography in 96-well filter plates markedly increased the total number of unique peptides and proteins that could be observed in SELDI–TOF mass spectra. Replicate fractionations generated nearly identical pH fractions, not only in terms of peptide and protein composition but also in respect to the ion signal intensity of replicate SELDI–TOF mass spectra. The feasibility of this approach was tested with samples from retinol-sufficient and retinol-deficient rats. The comparative analysis revealed reduced levels of three proteins with molecular masses between 10 000 and 20 000 in plasma of retinol-deficient rats. These results demonstrate that plasma profiling by anion-exchange fractionation and SELDI–TOF–MS may be a promising surveillance tool to detect changes in nutritional status and whole body physiology.
Journal of Chromatography A.
