CORE--PROTEOMICS

Yeshiva University, New York NY

Investigators

Linked publications, trials & patents

Abstract

DESCRIPTION (provided by applicant): Recently renamed the Proteomics Shared Resource, this facility, established in 1988, provides protein analysis using a combination of technologies: mass mapping and identification of unknown proteins from databases after solution or in-gel digests, sequencing by Edman degradation or by tandem mass spectrometry; 2D gels; MALDI-TOF or electrospray ionization mass spectrometry of natural and recombinant proteins and candidate drugs, HPLC-MS identification of components of macromolecular complexes, and identification of sites of posttranslational modifications. The JASCO J-720 circular dichroism spectropolarimeter is used for studies of solution conformation, and following reactions of enzymes specific for chiral substrates. The facility carries out routine and specialized peptide synthesis. The latter includes, beyond synthesis of peptide antigens and substrates, structural quality peptides (both natural and 15N labeled) for NMR analysis, peptides with modified groups, peptide baits for affinity capture of macromolecular complexes, and peptide scans of virus polypeptides for immune response assays. Since 1996, the Facility has added 2D gel capabilities, mass mapping coupled to database searches for protein identification, hydrogen deuterium exchange coupled to mass spectrometry for probing protein dynamics, synthesis of long peptides suitable for structural analysis, and high sensitivity separations. Four mass spectrometers of higher resolution and sensitivity have been acquired within the last two years: a PE-Biosystems DE STR mass spectrometer with delayed extraction, reflectron, high voltage detector and collision cell; a Finnigan LCQ ion trap mass spectrometer with a nanospray source; a Sciex API-365 electrospray ionization, triple quadrupole mass spectrometer with microionspray source; and a PE-Biosystems Mariner ESI TOF mass spectrometer with microionspray source. High sensitivity HPLCs for both off-line and on-line (linked to mass spectrometry) work have also helped the step-up to subpicomole detection sensitivity of proteins and peptides. The IPGphor high voltage system and the two Multiphor II flatbed units have added the capability for two-dimensional (2D) gel electrophoresis using immobilized ampholines for the isoelectric focusing dimension. New image and data handling software for 2D gels is being added. An Affinity Sensors surface plasmon resonance unit and Beckman XL-A analytical ultracentrifuge have added new experimental approaches for understanding biomolecular interactions in protein assembly. As scientific staff has turned over, new scientists at higher levels of experience have been added to the Proteomics Facility, building on the core of senior staff.