Proteomics

Proteomics Core Facilities Description

The UNR Proteomics/Mass spectroscopy Facility is located in two 600 ft2 laboratories on the first floor of the Fleischmann Agriculture Building. Proteomics equipment includes a Bio-Rad Protean IEF System for isoelectric focusing. Bio-Rad produces pre-cast IPG (immobilized pH gradient) strips for use with this machine. Bio-Rad's IPG strips have been recognized by many investigators for their consistency and versatility. The IPG strips may cover either broad pH ranges (pH 3-10) or appropriate narrow ranges (pH 5-7, for example) for higher resolution. The second dimension, SDS-PAGE electrophoresis, is conducted with precast Bio-Rad gels using a Protean II XL cell. A Bio-Rad VersaDoc imaging system is used for a variety of visualization techniques including Coomassie and silver staining, fluorescent dye staining (Sypro Ruby, etc.), and phosphoproteins staining. Stained gels are analyzed using Bio-Rad PDQuest 2-D Analysis Software for Mac or PC. Spots identified are automatically excised using a Bio-Rad ProteomeWorks spot cutter. The MS facility includes the Genomic Solution Investigator Proprep liquid handling robot for automated protein digestion and sample preparation. The instrument is fully enclosed in a HEPA-filtered enclosure and is capable of simultaneously performing 392 in-gel sample digests of 100 fmol of total protein and spotting directly to the 4700 MALDI target using a Zip-Tip protocol.

Our current Mass Spectrometry facility has the following instrumentation: a Finnigan Polaris Q ion trap MS interfaced with a Finnigan Trace GC with chemical ionization and electron impact, and a direct insertion probe for non-GC sample introduction. This instrument is capable of full-scan MS and MS/MS capabilities with unit resolution throughout the mass range of 10-1000amu. We have also purchased a NIST library of 147,000 compounds for analyte identification. A Michrom Paradigm 4 HPLC with four biocompatible peek heads capable of 50-5000 ul/min splitless gradient flow and 0.05 to 50 ul/min split gradient flow and two ten port micro valves offers the ability to conduct optimized application specific methods such as 2D-LC, Nano Trapping, Column switching. A Thermo Finnigan LCQ Deca Plus with both standard ESI (electrospray) ionization and nanoESI. Interfaced to the Michrom Pardigm for on-line LC/MS or direct infusion for pure or fractionated samples. This unit has a mass range of 4000 daltons and utilizes the highly regarded SEQUEST software for interpreting raw MS-MS data into protein sequence information, and then submitting interpreted data to BLAST searches. The facility also includes a LC Packings Probot Micro Fraction Collector that is a robotic system for fractionation, spotting, and sample preparation. This unit collects on-line fractions from the Michrom Paradigm HPLC to the ABI MALDI-TOF/TOF target. The Applied Biosystems 4700 Proteomics Analyzer is a high throughput MALDI-TOF/TOF with a resolution of > 12,000 and a mass accuracy of +/- 5ppm internal calibration and a mass range of > 200 kDaltons. The center’s workflow and data management is performed using the Proteus LIMS system from GenoLogics.   

The mass spectrometry facility is operated by Dr. David Quilici, a UNR graduate who has ably maintained and operated our mass spectrometers for the past 5 years. Dr. Kathleen Schegg, who is in charge of our Protein Microanalysis (Edman sequencing and amino acid analysis) and Peptide Synthesis Core Facilities, also operates the separations part of the proteomics facility. Drs. Quilici and Schegg, and Dr. David A. Schooley (Director of the Facility), are all members of the Association of Biomolecular Resource Facilities (ABRF), a society composed of researchers involved in DNA and protein microanalysis and synthesis. BRIN provides funds for hiring technicians for Drs. Schegg and Quilici to meet the expanded workload anticipated when our Proteomics facility is set up. We also have funds to employ student interns with BRIN funds to assist in developing optimal sample handling protocols if necessary.