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

Oxidative stress is believed to contributeto tissue injury in a number of humandiseases including atherosclerosis,neurodegenerative disorders, asthma, and aging.In atherosclerosis, for example, the productionof reactive oxygen and reactive nitrogen speciesappears to play a role in thevariety of processes thatinjure the arterial wall.Specifically, these reactivespecies have been found toparticipate in a cascade ofevents that could contributeto the progression of thedisease, including modificationof low-density lipoprotein(LDL), initiation of lipidloading into macrophages andsmooth muscle cells, cellproliferation and migration,and damage to the endothelium.The research activitiesin my laboratory are focusedon attaining a better understandingof the molecularmechanisms of oxidant injurythrough two lines ofinvestigation: characterizingthe anti-oxidant defensesystems in cells, and characterizingthe sites and chemicalstructure of oxidant damage to proteins.A unique aspect of these experiments isthe utilization of mass spectrometric methods ofprotein characterization and sequencing.The first area of investigation in thelaboratory uses a proteomic approach (2Delectrophoresis and tandem mass spectrometry)to map and identify the proteins that areoverexpressed and underexpressed in cell linesthat are resistant to oxidative injury. The generalhypothesis being tested in this work is that thesechanges in protein expression are responsible forthe resistant phenotype. As the differentiallyexpressed proteins are identified, subsequentexperiments will be designed and carried out totest their role in the injury process. Suchexperiments will use combinations of transfection,to increase protein expression, and antisenseoligonucleotide treatment, to inhibit proteinexpression, in combination with various in vitroassays of oxidant injury. The goal of this work isto discover previously unidentified and unstudiedThe Department of Cell BiologyProteomics and Mass Spectrometry TargetCharacterization of Anti-Oxidant DefenseMechanisms, Oxidative Protein Damage Sitesproteins that help cells resist the damaging effectsof oxidative stresses. In the longer term, we canthen begin to devise new methods to utilize theseproteins to intervene in diseases such as atherosclerosis.The second area of investigation in thelaboratory uses tandem massspectrometry to characterizethe site and structure ofoxidative modifications toproteins. One theory ofhow oxidative stress affectscells is that key proteinsbecome modified in amanner that alters theirfunction. The exact natureof these modifications,however, is not wellunderstood. It is envisionedthat the specific structuresthat are detected andcharacterized will providenew information about theoxidation reactions leadingto those modifications.Further, we expect that asour understanding of thesite and structure ofoxidative modifications isadvanced, it will be possibleto identify new molecularmarkers to monitor the effects oxidative stress invivo. This work includes the development ofnovel, site-specific quantitative methods foroxidized proteins.Michael T. Kinter, Ph.D.THE KINTERLABORATORYPOSTDOCTORAL FELLOWSJ. Andrew Keightley, Ph.D.Belinda B. Willard, Ph.D.STUDENTSJames ConwayLemin ZhengKinter, M., and N.E. Sherman (2000) Protein Sequencing and Identification UsingTandem Mass Spectrometry. John Wiley & Sons, Inc. New York, NY.Willard, B.B., and M. Kinter (2001) Effects of the position of internal histidine residueson the collision-induced fragmentation of triply protonated tryptic peptides. J. Am.Soc. Mass Spectrom. 12:1262-1271.Ruse, C.I., Willard, B., Jin, J.P., Haas, T., Kinter, M., and M. Bond (2002) Quantitativedynamics of site-specific protein phosphorylation determined using liquid chromatographyelectrospray ionization mass spectrometry. Anal. Chem. 74:1658-1664.Chikamori, K., Grabowski, D.R., Kinter, M., Willard, B.B., Yadav, S., Aebersold, R.H.,Bukowski, R.M., Hickson, I.D., Andersen, A.H., Ganapathi, R., and M.K. Ganapathi(2003) Phosphorylation of serine 1106 in the catalytic domain of topoisomerase IIalpha regulates enzymatic activity and drug sensitivity. J. Biol. Chem. 278:12696-12702.Willard, B.B., Keightley, J.A., Ruse, C.I., Bond, M., and M. Kinter (2003) Site-specificquantitation of protein nitration using liquid chromatography-tandem mass spectrometry.Anal. Chem. 75:2370-2376.77