This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are interested in developing mass spectral methods to follow and uncover chemistry of biological materials. We wish to do this 'in-situ;' that is, in the process of conducting MALDI or ESI. Free radical damage is one area where considerable inferential evidence points to the consequences of free radicals in damaging DNA and proteins. We believe that direct evidence of reactivity of biological material with free radicals could be gained by conducting the damaging chemical reactions in the electrospray source. To this end, we are constructing a special off-line ESI source where free radicals can be sprayed together with peptides and proteins. Following collection of the spray, we plan to analyze the 'spot' by either a second ESI experiment, coupled with MS/MS, or by MALDI and either PSD or MS/MS. Preliminary data show that the approach is feasible and that we are able to generate and react OH radicals with various peptides and small proteins. We intend to continue this developm ent as part of a general strategy to use contemporary ionization methods in MS as means for determining chemical and biochemical reactivity and as means of following interactions and conformation change. We are also considering a device incorporating vortex flows to help conduct the reactions and also to better focus the ions produced by electrospray.
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