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ABRF 2001 ABSTRACTS P41-M Review of gel- and column-based separation technologies for the identification of yeast proteins using MS/MS. V.C. Wasinger, G.L. Corthals; Garvan Inst. of Med. Res., 384 Victoria St., Sydney, NSW 2010, Australia Proteome analysis involves the identification and quantitation of expressed proteins by a given cell type, tissue or organism and most commonly involves 2-dimensional electrophoresis (2-DE) followed by mass spectrometry (MS) for protein separation and identification. While 2-DE is unequalled in its ability to separate and resolve thousands of proteins in complex solutions, we have recently shown that many proteins of biological significance lay beyond the detection limits of 2-DE. The application of 2-D gels becomes ineffective when proteins co-migrate to the same grid coordinates, merge to become one large spot or are not displayed because their concentration is simply too low for visualisation and subsequent biochemical MS analysis. Two important biological aspects prevent us from fully exploiting the power of the 2-DE technology: 1) Firstly, protein expression exists for up to 12 orders of magnitude (in serum). This broad range of expression critically limits the current 2-DE approach, as at best only 5 orders of magnitude difference in expression can be displayed; 2) “functional” proteins operate in association with other proteins; such that research must be directed toward the analysis of multiply interacting proteins or functional modules that regulate complex biological networks and pathways. Alternative fractionation methods such as chromatography exist and allow proteins/peptides to be separated based on similar (charge, size) or alternative (affinity, hydrophobicity) properties to 2-DE. Used in series with MS, these technologies have the potential to fractionate large numbers of proteins and peptides to enable the comprehensive, comparative and relational analysis of proteins. Yeast is an ideal choice for this study as both genome, proteome and transcriptome data is available for evaluation. A comparison of the separation potential based on protein numbers, protein classes and properties are described using 2-D gel electrophoresis, size exclusion and cation exchange chromatography followed by protein identification using ESI-MS/MS. P43-S Characterization of differential protein expression between wild type and Rce1 knockout mouse embryonic fibroblasts using 2-D SDS PAGE and MALDI TOF mass spectrometry. S.C. Hall1, D. Smith-Beckerman2, M. Lobo2, S.G. Young3; 1Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, 2San Francisco State Univ., 3UCSF Ras proteins play an important role in transmitting growth signals from membrane receptors to the nucleus, triggering the transcription of genes involved in cellular proliferation. Ras proteins are membrane bound, guanine nucleotide-binding proteins with GTP-ase activity. Many human cancers contain mutationally activated Ras proteins that transmit growth signals in an uncontrolled manner, triggering neoplastic transformation and uncontrolled cellular proliferation. The proper intracellular location of the Ras proteins depends on a series of posttranslational modifications: isoprenylation of a Cterminal cysteine, endoproteolytic release of the carboxyl-terminal three amino acids, and methyl esterification of the carboxyl-terminal isoprenylcysteine. Each of these processing steps represents a potential target for cancer therapy. The endoproteolytic cleavage step is carried out by the product of the Rce1 gene. Recently, the Rce1 gene was inactivated in mice, completely blocking the endoproteolytic processing of the Ras proteins. We used 2D-PAGE to assess differences in protein expression in primary embryonic fibroblasts from wild-type and Rce1 knockout mice. First, we wanted to detect substrates for Rce1. Altered electrophoretic mobility of individual protein spots on the gel indicated the possibility of aberrant post-translational processing. Second, we wanted to detect proteins whose expression level was affected by the Rce1 knockout mutation. MALDI TOF mass spectrometry was used to generate peptide mass fingerprints to identify several proteins having identical molecular weight, pI, and relative abundance in both wildtype and Rce1 knockout mice. Additional confirmation of the identities of these proteins was obtained by performing post-source decay analysis on selected tryptic peptides. It was important to identify these “marker” proteins as they will be used as migration reference points when comparing future 2D-gel separations. Furthermore, they permitted optimization of protocols for the MS analysis of Ras proteins from wild-type and Rce1 knockout fibroblasts. POSTER ABSTRACTS 198 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 P42-T Quantitative analysis of tumor antigens by mass spectrometry. A. Kishiyama, D. Arnott; Genentech, 1 DNA Way, South San Francisco, CA 94080 Both genomics and proteomics are useful for comparing gene expression patterns. The discovery of receptors overexpressed on tumor cells has lead to effective cancer therapies, for example. Techniques such as DNA arrays can be used to measure mRNA levels with great sensitivity and speed. More detailed information can be obtained through 2D gels, western blotting, and immunohistochemistry, but these protein-based approaches are time-consuming, prone to biases, or require generating antibodies. A method has therefore been developed, called the mass western experiment in analogy to the western blot, to better bridge genomics and proteomics. In this experiment, a variation on the ICAT experiment described by Gygi et al. (Nature Biotech. 1999 v.17 p.994), specific proteins (such as those found to be of interest from DNA array experiments) are detected and compared between samples. Proteins extracted from two samples are labeled with a custom ICAT reagent. The samples are mixed, digested, and the labeled peptides collected. LC-MS/MS is performed on an ion trap instrument; anticipated tryptic peptides from the protein of interest are continuously subjected to CID. Heavy and light ICAT-labeled peptides are simultaneously trapped and fragmented. Both identification and quantitation is thus obtained in one experiment. This approach has been validated by the comparison of cell lines expressing known tumor antigens in different amounts. Overexpression of the receptor Her-2 in breast cancer cell lines was shown, and by factors in agreement with other measurements. Other potential tumor antigens have likewise been detected. P44-M Intelligent data acquisition and automated sample analysis via orthogonal MALDI- QqTOF, a new tool for protein identification. C.M. Lock; MDS-Sciex, 71 Four Valley Drive, Concord, Ontario L4K 4V8, Canada The application of a novel UV-MALDI ionisation source coupled to an Applied Biosystems/MDS-Sciex QSTAR Pulsar QqTof mass spectrometer for protein sequencing and identification is described. The coupling of these two devices enables collision induced dissociation spectra of singly charged MADLI ions to be generated, with all the associated QqTof benefits of high mass accuracy and resolution. The inherent pulsed nature of the o-MALDI source is converted into a pseudo continuous beam of ions by collisional cooling in the Q0 region. The o-MALDI source is completely decoupled from and has no influence on the orthogonal Tof analyser. High mass accuracy and resolution is thus maintained simultaneously over the full mass range when switching between MS and MS/MS modes as opposed to conventional MALDI post source decay experiments. The application of the technique to the analysis of low femtomole unseparated protein tryptic digests is demonstrated using an automated data acquisition approach. The software developed enables the intelligent acquisition of data from sample plates with minimal user intervention. The high speed data acquisition capabilities of the o-MALDI source in combination with the high performance of the QqTof offers unique possibilities for rapid identification of proteins. Rapid analysis and identification of proteins via a peptide-mass fingerprinting approach and MS/MS sequence information will be shown.
P45-T Identification of changes in protein expression in Deinoccocus radiodurans using isotope-coded affinity tags. E.A. Panisko1, T.P. Conrads1, T. Veenstra1, L. Pasa-tolic1, G.A. Anderson1, R. Aebersold2, R.D. Smith1; 1Pacific Northwest Natl. Lab., P.O. Box 999, K8-98, Richland, WA 99352, 2Univ. of Washington The isotope-coded affinity tag (ICAT) strategy was used to identify the effects of irradiation on relative protein abundances in the highly radioresistant organism, Deinococcus radiodurans (D. radiodurans). The ICAT strategy uses two distinct isotopic versions of a cysteine-specific reagent; a “light” version and a “heavy” version in which eight hydrogen atoms are substituted for deuterium atoms. Proteome samples extracted from the organisms just prior to, as well as 30 min. and 3 hours after treatment with ionizing radiation were labeled with either the heavy or light isotopic version of the ICAT reagent. After mixing the samples in various combinations the cysteine containing polypeptides (Cys-polypeptides) were extracted using immobilized avidin chromatography. The Cys-polypeptides were initially identified by capillary reverse phase liquid chromatography (LC) using a conventional mass spectrometer (MS) operating in the tandem mass spectrometry (MS/MS) mode. The masses of the identified peptides were subsequently identified in a capillary LC separation coupled on-line with Fourier transform ion cyclotron resonance (FTICR) MS. Changes in relative protein expression were measured using the results obtained by LC/FTICR. The results show that most proteins undergo a decrease in expression when D. radiodurans is subjected to ionizing radiation. P47-M Use of stable isotope amino acid labels to simplify MS/MS peptide analysis. S.J. Berger, S-W. Lee, Y. Shen, G.A. Anderson, R.D. Smith; Pacific Northwest Natl. Lab., 902 Battelle Blvd. Box 999 MS: K8-98, Richland, WA 99352 Tandem mass spectrometry (MS/MS) is a primary analysis tool for the identification of peptides from proteolytic sample digests. When coupled with online separations, one or more peptides from a single 2D-gel spot digest can uniquely identify a given protein, or more complex digests can yield a global view of the proteome. The identification of individual fragment ions from a parent peptide is indirect, and the resulting peptide identification is strictly correlative. The ability to add sequence and composition constraints to assign peptide identity should significantly narrow the possible assignments of ions, and simplify interpretation of MS/MS spectra. Here we describe an application of stable isotope labeling in conjunction with MS/MS analysis of proteolytic digests that permit significant improvements assigning ions in the resultant MS/MS spectrum. Auxotrophic cells grown in parallel in the presence of a labeled or unlabeled amino acid are combined prior to cell disruption. Peptides derived from proteolytic digests generate pairs of peaks separated by a known mass difference during an initial LC/MS scan. These identified peptide pairs are isolated, and analyzed by LC/MS/MS in subsequent scans. When using a protease that cuts adjacent to the labeled amino acid, the paired/unpaired daughter ion pattern permits simplified identification of the parent peptide. This work was supported by OBER (U.S. DOE) and PNNL Laboratory Directed R&D. Battelle Memorial Institute operates PNNL for the U.S. DOE under Contract DE-AC06-76RLO 1830. POSTER ABSTRACTS ABRF 2001 ABSTRACTS P46-S Quantification of neurosteroids using NCI GC/MS. R.L. Fitzgerald; VA Med. Ctr., UCSD, VAMC-113, 3350 La Jolla Village Dr., San Diego, CA 92161 Electron capture negative chemical ionization (NCI) is one of the most sensitive ionization techniques available and is especially well suited for quantitative analysis of target compounds in biological extracts. For the analysis of small molecules, NCI is often several orders of magnitude more sensitive than traditional techniques such as electron ionization or positive chemical ionization. There are several important prerequisites for performing quantitative analysis using NCI, including an electronegative functional group, stable isotopic internal standards, and good method validation. We synthesized deuterium labeled analogs of neurosteroids and developed a NCI GC/MS method for quantification of neurosteroids in biological samples using isotope dilution. Neurosteroids have distinct neurotransmitter mediated effects and consequently it is important to be able to identify and quantify individual compounds. Previously, the determination of neurosteroids in biological matrices involved complicated purification protocols or did not use appropriate internal standards. We added deuterium-labeled internal standards to brain (100 mg of cortex homogenate) or plasma (300 �L). Samples were homogenized in methanol, centrifuged and diluted to contain 5% methanol and then applied to C-18 columns. After washing the column with methanol/water (50/50), steroids were eluted with methanol. Following evaporation, steroids were converted to pentafluorobenzyl oxime/trimethylsilyl ether derivatives. The extracts were analyzed using SIM. The present method allows simultaneous quantification of pg amounts (100 pg in 300 �L of plasma and 250 pg in 100 mg of brain tissue) of neurosteroids and will be helpful in elucidating the role of neurosteroids in health and disease. P48-T A comprehensive proteomic analysis of human cilia using nanoscale capillary LC/MS/MS. M. Moyer1, K. Blackburn1, W. Burkhart1, A. Moseley1, L. Ostrowski2, R. Boucher2; 1Glaxo Wellcome, 5 Moore Drive, Research Triangle Park, NC 27709, 2Univ. of North Carolina Ciliated cells play an integral role in the defense mechanisms of the respiratory system. By the coordinated beating of their cilia they provide the force necessary to clear potentially harmful material from the airways. In order to better understand the protein composition of cilia, human cilia were subjected to a comprehensive proteomic analysis. Cilia were isolated from cultures of airway epithelial cells and component proteins separated by 1D or 2D gel electrophoresis. Bands or spots were excised, subjected to in-gel proteolytic digestion, and component proteins identified by nanoscale capillary LC/MS/MS. Alternatively, proteolytic digests of intact cilia were analyzed directly by nanoscale capillary LC/MS/MS with or without the use of cysteinespecific affinity tags. Data will be presented on proteins identified as components of cilia as well as details of the analytical methodologies. JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 199
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