The Lerner Research Institute's Mass Spectrometry Laboratory for Protein Sequencing is a proteomics laboratory that uses advanced tandem mass spectrometry methods for the sequencing and identification of proteins in electrophoretic gels. The goal of the laboratory is to make advanced 2D electrophoresis mapping and mass spectrometric protein and post-translational identification and quantitation methods available to all investigators at the Cleveland Clinic.
The most common experiment that is performed is to cut protein bands from Coomassie blue-stained gels and digest that protein directly in the gel band. Any protein that can be detected by Coomassie blue staining can be sequenced by our methods. The same experiment can also be carried out with silver-stained gel bands provided the silver staining protocol does not include glutaraldehyde in the gel fixing steps. Silver stained gels, however, are far more susceptible to contamination by background proteins such as keratins, making successful analyses far less reliable.
Other types of mass spectrometric experiments can be considered. In general, however, experiments that do not involve proteins and peptide will not be compatible with the instrument conditions that must be preserved for the protein sequencing experiments.
All investigators considering the use of mass spectrometric sequencing experiments are encouraged to contact Dr. Belinda Willard at 444-7170.
The major equipment in the Proteomics laboratory includes two capillary column HPLC-electrospray ionization-tandem mass spectrometry systems and one MALDI-TOF system.
The electrospray systems include a ThermoFinnigan LCQ-deca that was purchased by the Cleveland Clinic, and a ThermoFinnigan LTQ that was purchased in 2004 with funding from the Ohio Board of Regents Hayes Investment Fund as a part of the Ohio Mass Spectrometry Consortium, The MALDI-TOF mass spectrometer is a Micromass TofSpec 2E that was purchased in 2001 by the Cleveland Clinic. All of these instruments are dedicated, by necessity, to high-sensitivity protein sequencing and identification experiments.
In addition, the laboratory routinely uses a 3-node Mascot cluster for the data base searches. We also have several stand-alone copies of Sequest and a copy of Sieve for added computational capabilities. Finally, the laboratory also contains a variety of electrophoresis equipment, including systems for isoelectric focusing from both Pharmacia and BioRad, and appropriate SDS-PAGE systems, with scanning equipment, computer facilities, and software (PDQuest) for the analysis of the 2D gels that are produced.
Lerner Research Institute
Cleveland Clinic, Mail Code NB21
9500 Euclid Avenue
Cleveland, Ohio 44195
