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. Differential isotopic labeling of peptides has been used for quantitative proteomics. Beta-elimination/michael addition has been used for mapping of ser/thr post-translational sites and derivitization of cysteines. We are Developing a method termed """"""""BEMAD"""""""" which combines these applications for the purpose of simultaneous identification and quantitation of both expression and ser/thr post-translational changes in proteomics. In this method, we are optimizing beta-elimination and addition of """"""""light"""""""" DTT vs. """"""""heavy"""""""" (deuterated, d6) DTT to formerly phosphorylated or O-GlcNAc modified serines and threonines in a manner which can discriminate between these alternative modifications. Additionally, we are developing methods for the application of this chemistry to derivitizing cysteines for expression proteomics. We are in the process of modifiying public search algorithms for interpretation of MS/MS such as MASCOT and Protein Prospector such that the variable of DTT addition to either serine, threonine, or cysteine can be incorporated into the search, and so that the ratio of ion pairs modified by either light or heavy DTT can be automatically quantitated. This method should be widely applicable to comparitive proteomic studies. (Additional effort and instrument time reported under Collaborative projects and other Technical Research and Development projects.)
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