Transient Intermediates in Proton and Electron Transfer

University Of Washington, Seattle WA

Investigators

Abstract

In this project funded by the Chemical Structure, Dynamic & Mechanism B Program of the Chemistry Division, Professor Frantisek Turecek of the Department of Chemistry at University of Washington, Seattle, will develop new methods for improved protein sequencing by mass spectrometry. The goal of this research is first to gain deep understanding of peptide backbone dissociations that provide information on the peptide amino acid sequence which is then spliced to complete the sequence of the parent protein. Protein sequencing is the critical step in determining mutations, post-translational modifications, and damage that cause many diseases and degenerative processes. Cancer, Parkinson and Alzheimer diseases and aging all include protein changes as one of the major causes disrupting cellular processes and their regulation. This research group is also well-positioned to provide the highest level of education and training for students of different cultural backgrounds including those underrepresented in physical and life sciences. Mass spectrometry is the method of choice for protein and peptide analysis. In the so-called bottom-up approach, the protein or a mixture thereof is enzymatically cleaved to a mixture of peptides that are separated on-line and analyzed. Mass spectrometry analysis provides valuable information on the peptide mass and amino acid sequence and composition. The information can be evaluated by matching against a data base to find the generic protein for which there is a known gene. However, proteins from organisms lacking genome data, or modified proteins due to disease or damage must be analyzed by the so-called de novo sequencing, implying complete sequence readout from the mass spectrometry data alone. In this project, new methods are investigated and developed to improve de novo protein sequencing. The project has several goals: (1) A new method of peptide capture and chemical modification on solid phase support is being developed to simplify peptide mixtures and increase the level of sequence readout. (2) Fundamental studies of peptide ion dissociations following electron transfer will be carried out to determine the effects of ion 3D structure and energy deposition. (3) A new UV photodissociation method is introduced to provide a different means of activation, causing efficient dissociation of gas-phase peptide ions. Results from these studies will be used to refine the current theoretical model of peptide ion activation and dissociation and utilize it for prediction of ion behavior.