The goal of this proposal is to develop and characterize non-enzymatic based methods for single-step cell lysis and site-specific protein digestion. It is expected that the developed techniques will simplify sample preparation and increase sample throughput in proteomic related measurements. Moreover, the ability to prepare the sample by non-enzymatic means has the potential to yield simpler and faster methodology that can be incorporated into proteomic-based lab-on-a-chip devices. Toward these goals, two methodologies will be developed in this work, and include electrochemical and microwave-heating chemically induced reactions at specific amino acids. In this proposal, the development and testing of these methodologies will be carried out with a focus on the detection and identification of microorganisms (i.e., pathogens). To achieve this, the following studies will be carried out: (i) Characterization and optimization of digestion parameters for each method studied using model peptides and proteins for site specificity and possible side reactions, (ii) Test each methodology with model microorganisms for their ability to lyse the cell and produce peptides suitable for MS/MS analyses, and (iii) Develop a prototype lab-on-a-chip device (electrochemical method only) to conduct the lysis/digestion step on-line with MS/MS detection. Specifically, each method will be investigated for its ability to induce cell lysis and digestion of its proteins, all in a single step. The long term goal of this project is to develop and characterize novel protein chemistry that can be incorporated into simple lab-on-a-chip devices for sample pre-processing prior to MS-based proteomic measurements. Advantages anticipated from these studies include the ability to perform high throughput sample preparation for proteomic measurements and its direct effect on the rapid diagnosis of microbial related diseases. ? ?
