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RP Chromatography (rp + chromatography)
Selected AbstractsA fully automated 2-D LC-MS method utilizing online continuous pH and RP gradients for global proteome analysisELECTROPHORESIS, Issue 23 2007Hu Zhou Abstract The conventional 2-D LC-MS/MS setup for global proteome analysis was based on online and offline salt gradients (step and continuous) using strong-cation-exchange chromatography in conjunction with RP chromatography and MS. The use of the online system with step salt elution had the possibility of resulting in peptide overlapping across fractions. The offline mode had the option to operate with continuous salt gradient to decrease peak overlap, but exhibited decreased robustness, lower reproducibility, and sample loss during the process. Due to the extensive washing requirement between the chromatography steps, online continuous gradient was not an option for salt elution. In this report, a fully automated, online, and continuous gradient (pH continuous online gradient, pCOG) 2-D LC-MS/MS system is introduced that provided excellent separation and identification power. The pH gradient-based elution provided more basic peptides than that of salt-based elution. Fraction overlap was significantly minimized by combining pH and continuous gradient elutions. This latter approach also increased sequence coverage and the concomitant confidence level in protein identification. The salt and pH elution-based 2-D LC-MS/MS approaches were compared by analyzing the mouse liver proteome. [source] The application of novel 1.7 ,m ethylene bridged hybrid particles for hydrophilic interaction chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 9 2008Eric S. Grumbach Abstract An un-derivatized 1.7 ,m ethylene bridged hybrid (BEH) particle was evaluated for its utility in retaining polar species in hydrophilic interaction chromatography (HILIC), and was compared to a 3 ,m un-derivatized silica material. Retentivity as a function of mobile phase pH, polar modifier and ACN content was examined. Also, the efficiency of the two particle substrates was compared by plotting HETP vs. linear velocity. Improved chemical resistance of the un-derivatized BEH particle was compared to un-derivatized silica at pH 5, demonstrating no performance deterioration over the course of 2000 injections for the BEH particle, while the silica particle deteriorated rapidly after 800 injections. Lastly, ESI-MS sensitivity as a function of particle size and separation mode was demonstrated. A 2.2 to 4.7-times higher ESI-MS response was observed on the 1.7 ,m particle compared to the 3 ,m particle, whereas a 5.6 to 8.8-times higher ESI-MS response was observed using HILIC as when compared to traditional RP chromatography. [source] Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharidesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2008Johannes Stadlmann Abstract Two LC-ESI-MS methods for the analysis of antibody glycosylation are presented. In the first approach, tryptic glycopeptides are separated by RP chromatography and analyzed by ESI-MS. This "glycopeptide strategy" allows a protein- and subclass-specific quantitation of both neutral and sialylated glycan structures. Additional information about under- or deglycosylation and the protein backbone, e.g., termini, can be extracted from the same data. In the second LC-ESI-MS method, released oligosaccharides are separated on porous graphitic carbon (PGC). A complete structural assignment of neutral and sialylated oligosaccharides occurring on antibodies is thereby achieved in one chromatographic run. The two methods were applied to polyclonal human IgG, to commercial mAb expressed in CHO cells (Rituximab, Xolair, and Herceptin), in SP2/0 (Erbitux and Remicade) or NS0 cells (Zenapax) and the anti-HIV antibody 4E10 produced either in CHO cells or in a human cell line. Both methods require comparably little sample preparation and can be applied to SDS-PAGE bands. They both outperform non-MS methods in terms of reliability of peak assignment and MALDI-MS of underivatized glycans with regard to the recording of sialylated structures. Regarding fast and yet detailed structural assignment, LC-MS on graphitic carbon supersedes all other current methods. [source] Global phosphoproteome analysis on human HepG2 hepatocytes using reversed-phase diagonal LCPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2005Kris Gevaert Professor Abstract We present a phosphoproteomics approach using diagonal RP chromatography as the basic isolation principle. Phosphopeptides present in a tryptic digest of total cellular lysates were first enriched by Fe3+ -immobilized metal ion affinity chromatography. Further sorting of the phosphopeptides took place in three steps. First, the resulting peptide mixture was fractionated over reversed-phase chromatography. Second, peptides present in each fraction were treated with phosphatases. Third, the dephosphorylated peptides were then more hydrophobic and shifted towards a later elution interval from the contaminating non-phosphopeptides eluting at the same position as during the primary run. Since the phosphopeptides are isolated as their dephosphorylated form, additional proof for their original phosphorylation state was obtained by split-differential 16O ,18O labeling. The method was validated with alpha-casein phosphopeptides and consecutively applied on HepG2 cells. We identified 190,phosphorylated peptides from 152,different proteins. This dataset includes 38,novel protein phosphorylation sites. [source] | ||||||||||