PSMeeting_Philadelphia_12-03

Title	Abstract
"Comprehensive Analysis of Complex Proteomes Using 3-D and 4-D Protein Profiling Strategies" David W. Speicher, The Wistar Institute, speicher@wistar.upenn.edu	No current protein profiling method is capable of quantitatively comparing more than a few thousand proteins at a time. Hence, it is necessary to prefractionate samples if more comprehensive coverage of complex proteomes such as human cells and biological fluids is desired. Of all the alternative methods available for prefractionating proteins, preparative scale solution IEF methods yield the best separation between fractions with minimal cross contamination, and reproducible and high resolution separations are essential for meaningful quantitative comparisons. A solution IEF prefractionation method that is particularly well scaled for proteome analysis is MicroSol-IEF using the ZOOM IEF Fractionator. The resulting pools can be analyzed using: 1) narrow range 2-D gels; 2) LC/LC-MS/MS or so called MudPIT analysis; or 3) a novel 2-D batch array/pixelation approach. Each of these downstream separation methods has its strengths and weaknesses. The combination of ZOOM IEF prefractionation and slightly overlapping narrow range 2-D gels is particularly powerful and useful for projects involving comparison of more than two samples. We have been able to quantitatively analyze at least 5,000 to 10,000 proteins from human cancer cells and plasma with effective separation distances of more than 80 cm for the pH 3 to 10 range of a proteome. Another powerful profiling approach is the "Batch 2-D Array/Pixelation method". In this method, each ZOOM IEF fraction is separated for a short distance on a 1-D SDS gel and each lane is sliced into a uniform number of bands. The combined solution IEF and short SDS gel produce a 2-D array that circumvents many of the limitations of 2-D gels while retaining information concerning protein size and charge. Each point or pixel on this 2-D grid is analyzed by LC-MS/MS and results from all runs are combined to produce much more extensive proteome coverage than most alternative profiling methods for complex proteomes such as human cell extracts and biological fluids such as serum or plasma.
"Expending Dynamic Range of Human Serum Protein Detection and Identification by Affinity Removal of Multiple High-Abundant Proteins" Kelly Zhang, Agilent Technologies, Kelly_zhang@agilent.com	Given its important function in circulating and exchanging important molecules around the body, human plasma/serum has been utilized as a valuable source for the discovery of new biomarkers for disease or novel drug targets. A few well-characterized high-abundant proteins such as albumin, IgG, transferrin, haptoglobin, IgA and anti-trypsin represent up to 85 - 90% of the total protein mass in serum. These proteins have become a major barrier blocking detection of other proteins in this fluid by limiting the dynamic range of mass loading for protein analysis. Dye-based affinity chromatography (Cibacron Blue) for human albumin removal has been shown to incompletely remove albumin and nonspecifically remove many other non-targeted proteins. New technologies for specific and reproducible removal of these proteins from proteomics samples are needed to allow researchers to dive deeper into the human plasma proteome. The Agilent Multiple Affinity Removal System was developed to meet the critical requirements for proteomics sample preparation. An affinity column, packed with multiple immobilized polyclonal antibodies, combines with optimized buffers for minimizing non-target protein removal, provide a reliable and ready-to-use tool for proteomics scientists. The affinity column captures and removes simultaneously the six high-abundant proteins as mentioned above from human serum. The optimized buffers ensure efficient and reproducible removal of these proteins and effective column regeneration for at least 200 sample injections. Robust depletion of high-abundant proteins has been demonstrated by 1D and 2D gel patterns, MALDI-TOF, and LC/MS analysis. Superior specificity of depletion has been observed in comparison to Cibacron Blue when bound fractions were analyzed by in-gel trypsin digestion, followed by LC/MS/MS analysis. The column removes the masking effect of high-abundant proteins from proteomics samples such as plasma and other bodily fluids, increases mass loading of low-abundant proteins, thus expanding the dynamic range of current LC/MS, 1DGE and 2DGE analytical methods.
"Reducing Sample Complexity by Isoelectric Prefractionation: Comparison of Fractionation Devices" Gary B. Smejkal, Proteome Systems Inc., gary.smejkal@proteomesystems.com	Sample complexity frequently interferes with the analysis of low abundance proteins by two-dimensional gel electrophoresis (2DGE). Ideally, high abundance proteins can be removed allowing low abundance proteins to be applied at much higher concentration than in the unfractionated sample. One approach is to partition the sample in a manner that segregates the bulk of extraneous proteins from the protein(s) of interest. Preparative liquid phase IEF produces fractions of discrete pH intervals allowing isolated narrow segments of a proteome to be analyzed individually, thus providing a unique opportunity to visualize low abundance proteins by 2DGE. It is particularly useful for the isolation of low abundance proteins of extremely basic or acidic pI. Two such isoelectric fractionation devices, namely the Invitrogen ZOOM IEF Fractionator and the Proteome Systems multi-compartmental electrolyzer (MCE) were compared for the fractionation of Sacharomyces cerevisae cytosolic proteins prior to 2DGE.
"Proteomics Of Oxidative Stress" Ian Blair, University of Pennsylvania, ian@spirit.gcrc.upenn.edu	Oxidative stress results in the formation of reactive oxygen species (ROS), which can damage cellular macromolecules such as DNA and proteins. DNA and protein damage results directly from ROS, or from ROS-derived lipid-hydroperoxides that break down to form the alpha, beta-unsaturated aldehyde genotoxins, 4-oxo-2-nonenal, 4-hydroxy-2-nonenal, and 4,5-epoxy-2(E)-decenal. Lipid hydroperoxides are also formed enzymatically during oxidative stress from 5-lipoxygenase (5-LOX), 15-LOX, cyclooxygenase-1 (COX-1), and COX-2. Both 15-LOX and COX-2 convert linoleic acid into 13(S) hydroperoxyoctadecadienoic acid (13-HPODE) the prototypic n-6 polyunsaturated fatty acid (PUFA) hydroperoxide. We have investigated the homolytic decomposition of 13-HPODE with a variety of initiators. These studies characterized 4-hydroperoxy-2-nonenal, 4-oxo-2-nonenal, 4-hydroxy-2-nonenal, and 4,5-epoxy-2-decenal as lipid hydroperoxide-derived bifunctional electrophiles. We made the surprising observation that vitamin C can stimulate the breakdown of lipid hydroperoxides to alpha, beta-unsaturated aldehyde genotoxins. Using LC/electrospray (ESI)/MS/MS and matrix-assisted laser desorption/ionization/time of flight (MALDI/TOF)/MS and MALDI/TOF/MS/MS we have begun to systematically characterize the lesions that can also occur in proteins from these bifunctional electrophiles. Functional studies have focused on histone proteins because of the possibility that lipid hydroperoxide-mediated epigenetic effects may be induced during oxidative stress. Using a combination of deuterium isotope labeling and LC/MS/MS, it was demonstrated lipid hydroperoxides caused the formation of a cyclic peptide on the HAK motif of histone H4. Current experiments are directed at demonstrating whether this modification can occur on histone proteins during oxidative stress in vivo. Supported by NIH RO-1 CA95586 and RO-1 CA91016