The proposed research will develop a new technology for highly automated and highly sensitive analytical systems for the protein components of complex biological systems. The technology will combine multi-step sample processes in silicon chip formats with highly sophisticated tandem mass spectrometry analyses. Micromachining methods that have already been successful in producing chambers, channels, check valves, filters and electrospray emitters will be extended to include active valves and micro-pumps. The methodologies needed to place chemical systems within the chip-based structures to perform affinity isolations, enzyme digestions, and chromatographic and/or electrophoretic separations will be developed. By combining these operations in a single microscale device, the sample losses in going from one step to the next will be avoided. These chip-based sample preparation systems will be interfaced to ion trap mass analyzers capable of multiple stages of tandem MS analysis. Control of both the on-chip processes and the MS analysis will be accomplished using a single expert system program. This program has the capability of analyzing data in real time and changing the course of the analysis according to a predefined set of rules. The net result will be a much more efficient and comprehensive method of collecting data, thus minimizing the amount of time and sample needed. Applications of the new technology will focus on the characterization of protein posttranslational modifications with initial emphasis on sites of phosphorylation. The principal goal of this research is to package very sophisticated analytical tools and expertise in a form that can be readily exported to other laboratories.
