Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

MASS SPECTROMETRY CORE I: PROTEIN SEQUENCINGThe first Mass Spectrometry Core laboratory was created in June 1999 with the goal of makingadvanced methods and instrumentation for protein sequencing available to the investigators in theCleveland Clinic. The laboratory is equipped with a Finnigan LCQdeca ion trap mass spectrometrysystem, with a capillary column liquid chromatography inlet and a microspray ionization source, and aMicromass TofSpec 2E matrix-assisted laser desorption/ionization time-of-flight mass spectrometrysystem. These systems are able to acquire a variety of sequence-specific information that is used tosearch the growing protein and genome sequence databases to identify a protein.The key aspect of the protein sequencing and identification experiment is the simple fact that,because of the sensitivity of the experiment, any protein band that can be visualized in a Coomassie bluestained gel can be sequenced and identified. The analysis begins by cutting the protein band of interestout of the gel and digesting it directly in that piece of polyacrylamide. This digest produces a relativelylarge number of peptides derived from the protein that are sequenced in each analysis. As a result, theexperiment is direct, rapid, sensitive, and identifies the protein based on the characterization ofapproximately one half of its sequence. The sensitivity of the experiment also allows one to considersequencing proteins in silver-stained gel bands, but extra care must be taken to use compatible stainingtechniques and to avoid contamination of the gel with background proteins.The laboratory also has a variety of electrophoresis systems that may be of interest to investigators.These systems include 2D electrophoresis systems that use immobilized pH gradient strips for theisoelectric focusing and high resolution, pre-cast gels for the second dimension. These systems areavailable for the development of 2D methods for complex protein separation problems.MASS SPECTROMETRYCORE I:PROTEIN SEQUENCINGDIRECTORMichael Kinter, Ph.D.SUPPORT PERSONNELBelinda B. Willard, Ph.D.Andrew Keightley, Ph.D.Lemin ZhengWebsite: http://www.lerner.ccf.org/services/mass_spec/MASS SPECTROMETRY CORE II: MOLECULES/SMALL COMPOUNDSIn 2001, the Lerner Research Institute established a second Mass Spectrometry Core, functioningas both an investigative core and a service core research facility. The main focus of this core is quantificationof molecules in complex matrices and structural characterization of small compounds. The coreis designed to meet the growing needs of investigators to develop analytical methods for detection andquantification of biomarkers in plasma, tissue and other biological materials.The core is equipped with a Micromass Quattro Ultima triple quadruple mass spectrometry systemand a Beckman HPLC equipped with autosampler, photodiode array and fluorescence detectors. Themass spectrometer has two ionization sources available: electrospray ionization (ESI) and atmosphericpressure chemical ionization (APCI). The effective mass range is 4180 Da for singly charged species andhigher for multiply charged species. Reverse phase HPLC/MS or online HPLC tandem mass spectrometry(LC/MS/MS) analysis is available with this instrument.The MS II Core, under the direction of Dr. Stanley Hazen, was made possible by the award of aninstrument grant from the NIH. The core manager, Dr. Renliang Zhang, has both biochemistry andanalytical chemistry background and extensive experience with HPLC and mass spectrometry.MASS SPECTROMETRYCORE II:MOLECULES / SMALLCOMPOUNDSDIRECTORStanley Hazen, M.D., Ph.D.MANAGERRenliang Zhang, M.D., Ph.D.http://www.lerner.ccf.org/services/ms2/185