Events

Title	Abstract
"Phosphotyrosine Generated Signals in Squamous Carcinoma Analyzed by Affinity Chromatography and Mass Spectrometry" settinca@appliedbiosystems.com Tina Settineri, Applied Biosystems	Over-expression and/or enhanced activation of the epidermal growth factor receptor (EGFr) are frequently observed in human cancers, and EGFr over-activation correlates with poor prognosis. Affinity chromatography and mass spectrometry were used to define phosphotyrosine-containing protein complexes involved in EGFr signaling. The study used both capillary LC methods of protein (C4) and peptide fractionation (C18, SCX), and 1D SDS-PAGE methods. The model system used was HN5 squamous carcinoma cells over-expressing EGFr, both with and without treatment with the EGFr kinase inhibitor OSI-774 (Tarceva) for 1 hour prior to EGF for 10 minutes. Phospho-tyrosine (PY)-containing protein complexes were bound to immunoaffinity resins, washed with TBS and eluted with 0.1% TFA, 5% MeOH. Proteins were subjected to C4 reverse phase LC, SDS-PAGE or left unfractionated. Proteins were then reduced and acetamidomethylated prior to protease digestion with trypsin and then fractionated by C18 reverse phase or coupled SCX/C18 on-line (Applied Biosystems\|MDS Sciex QSTAR Pulsar). Protein complexes associated with EGFr signaling, internalization and degradation were observed.. 34,931 MS-MS attempts using a 2s survey scan followed by three 5 second product scans were refined to 1813 peptide sequences using stringent selection criteria (Pro ID Software; Applied Biosystems\|MDS Sciex). 34 individual EGFr peptides were sequenced from multiple MS-MS scans. 81 of 129 previously MALDI-assigned proteins were identified by IDA-LC-MS-MS using established cutoff criteria. While OSI-774 markedly reduced PY content within EGF treated HN5 cells, EGFr interactions with internalization-associated proteins appeared unperturbed. Nine proteins of unknown function were observed and novel proteins and complexes can be detected using this type of methodology. The differential ICAT labeling technique was used to measure protein changes associated with the blockade of EGFr kinase activity by the specific quinazoline inhibitor OSI-774 (Tarceva), showing marked redution in PY content as a result of Tarceva treatment.
"Proteomics In Reproductive Medicine: Molecular Signatures of PPROM" lubi@ucsd.edu Ljubica Bogic, UCSD	Pre-term premature rupture of fetal membranes (PPROM) is a pregnancy disorder with a multi-factorial etiology. Due to its complexity, the molecular and cellular events underlying the disease are still unknown. Key molecular features of this disorder lie in abrogated events at the amnion-chorion/decidua interface. Research in the pathologies of infection-mediated pre-term birth and thus PPROM has historically focused on the regulation of intrinsic factors, however it has become evident that extrinsic factors (e.g., the local stroma microenvironment) are equally important mediators of host cell response to pro-inflammatory signals. The multifaceted cellular responses to extracellular matrix (ECM) organization, specifically cell-cell and cell-ECM interactions and tissue remodeling, demonstrate that the mechanism of PPROM can no longer be defined without taking into account extracellular regulators of normal and pathologic cellular behavior, cell-cell/cell-ECM communication and ECM collagenolysis. It is likely that the multiple factors control matrix metalloproteinases activity in fetal membrane tissue and finally lead to their rupture during pregnancy belong to biologically active molecules of stroma, suggesting cell-ECM co-operation. In addition, proteolysis of ECM components by matrix metalloproteinases (MMPs) can alter these functions by the release of ECM-fragments with distinct biological activities. Thus, these various components of fetal membrane rupture pathway need to be considered when trying to determine the key events regulating matrix collagenolysis and hence PPROM. In order to reveal unique and specific protein signatures of prematurely ruptured fetal membranes (PROM) that eventually could predict its clinical phenotypes (preterm vs term), we used Ciphergen Biosystem’s ProteinChip® Arrays and surface-enhanced laser desorption/ionization (SELDI) technology in a global evaluation of the human fetal membrane tissues at the protein level.
"Proteomic Characterization of Human Pathogens " Sandra McCutchen-Maloney (replaced Gloria Murphy), LLNL smaloney@llnl.gov	It is clear that pathogens still pose a significant threat to human health both through natural environmental exposures and through acts of bioterrorism. The Biodefense Proteomics Group at Lawrence Livermore National Laboratory is interested in characterization of Yersinia pestis, the causative agent of plague. Although changes in gene and protein expression have been linked to virulence, the critical proteins, pathways and mechanisms are in general still poorly understood. To identify alterations in protein expression resulting from induction of the Y. pestis virulence mechanism, we are using multidimensional protein separation techniques followed by mass spectrometry. To probe the protein interactions critical to virulence, we have developed a SELDI-MS method to define protein-protein interactions within the pathogen and between the pathogen and the human host. This work provides mechanistic details regarding Y. pestis virulence and pathogenicity as well as host response, and ultimately may define biomarkers for detection of plague as wells as novel therapeutic targets.
"Profiling and Identification of Human Serum Proteins and Metabolites: A Clinical Approach to Proteomics" mroy@surromed.com Sushmita Roy, Surromed, Inc.	The ability to profile cells, proteins and metabolites in human blood reliably and in depth, and compare profiles of normal, diseased and medicated populations, offers a path to biomarker discovery. SurroMed uses its integrated phenotyping and biological marker discovery platform in clinical studies focused on understanding disease presentation, progression and response to therapy. Our biomarker discovery platform incorporates advanced proprietary technologies for profiling and analysis of hundreds of immune cell populations, proteins and metabolites. Towards the goal of biomarker discovery, we have developed a high-throughput proteomics/metabolomics platform that features reproducible serum sample processing, quantitative mass spectrometric analysis, automated clinical data analysis and protein identification. The SurroMed proteomic platform is based on minimal fractionation and nano-flow liquid chromatography coupled to mass spectrometry. Human proteins are separated from the metabolome, digested peptides are separated by nano-flow reverse-phase liquid chromatography and then mass analyzed at high resolution (time-of-flight instrument, LCT, Micromass). When peptides are observed to undergo concentration changes between samples, they are then identified by combining the accurate parent mass (m/z with charge state) provided by the time-of-flight instrument with an MS/MS spectrum provided by an ion trap mass spectrometer (LCQ Deca, ThermoFinnigan). Quality control studies show that our sample processing and analysis methods are efficient and reproducible. Typical average coefficient of variation (CV) for large numbers of peaks are ~ 25% from sample to sample. The mass spectrometric profiles of serum samples from individuals using only 20 micrograms of final protein (equivalent to ~ 5 microliters of human blood) show several thousand components. To date, over 400 proteins represented by a thousand unique peptide sequences have been identified and tracked. We are able to fully analyze 50 samples a week per instrument in a robust manner, pick and quantify peaks in an automated fashion and mine the clinical data sets in search of biomarkers. Several large clinical studies are in progress in asthma, allergy, diabetes and rheumatoid arthritis. Disease related proteins and their changes will be discussed.