Proteomics Core

Instruments

Isoelectric Focusing and SDS-PAGE

The MSU Proteomics Facility utilizes the ProteomeWorks system from Bio-Rad consisting of an FX Pro Plus molecular imager and ProteomeWorks spot cutter complete with PDQuest spot identification software. We utilize two gel sizes for 1D and 2D-PAGE; Criterion (11 cm IEF) and Protean II (17 cm IEF) and various pH ranges and precast gel types are available. Gels can be stained with either Colloidal Coomassie, Krypton fluorescent or mass spec compatible Silver stains. Additionally, the facility offers protein and peptide separations in the liquid phase using the Agilent 3100 Offgel Fractionator or the Invitrogen ZOOM IEF Fractionator. Using the Offgel Fractionator proteins/peptides can be separated into 12 or 24 distinct fractions over various pH ranges. For larger protein loads, the ZOOM IEF fractionator can be used to generate up to 7 pH fractions between pH 3 and 12. Any of these fractions can be further used for second dimension SDS-PAGE separations or desalted and analyzed directly on a mass spectrometer.

LC/MS/MS

The MSU Proteomics Facility contains three state of the art mass spectrometers. The workhorses of the facility are two ThermoScientific LTQ Linear Ion Trap mass spectrometers which offer unparalleled speed, sensitivity and MSn spectral quality for protein identification, both large and small scale. For more specific MS/MS experiments we also operate a ThermoScientific LTQ-FTICR Ultra hybrid mass spectrometer capable of sub p.p.m. mass accuracy and peak resolutions greater than 750,000. The recently upgraded Ultra cell and ECD source on the LTQ-FT produces enhanced sensitivity and greater dynamic range allowing unprecedented analytical power and versatility. Each one of these mass spectrometers is coupled to a Waters nanoAcquity UPLC which is optimized for high-resolution identifications and separations at precise nano-flow rates and outfitted with a MICHROM Bioresources ADVANCE nano-spray source. Additionally, a standalone Dionex p680 HPLC is available for SCX or HILIC fractionation of complex protein samples prior to MS analysis.

Peptide spectral matching is performed using the Mascot searching algorithm licensed to an actively managed in-house server. Post-translational modification searches can be performed using either Mascot or PEAKS de novo sequencing software. Results are further validated using the Scaffold software package.