The determination of biological processes necessary to provide a detailed understanding of diseased states requires a comprehensive view of both the genome (DNA) and proteome (proteins). There have been major advances in genome analysis; however, technology for study of the proteome has lagged behind. The overall goal of our research program is to develop new technology to enhance the throughput and quality of information available in the analysis of the proteome. Our vision is to design an integrated instrument that will identify approximately 1000 proteins per hour and characterize 200-300 proteins per hour with mass detection limits of less than 1 fmole. This throughput would allow complete analysis of a 2-D gel separation of a cell lysate in one day. To achieve this goal, the proposed work would involve the construction, for the first time, of a multiplex capillary array matrix assisted laser desorption time-of-flight (MALDI/TOF) mass spectrometer (MS). Our laboratory has already demonstrated the feasibility of on-line capillary infusion and separation-MALDI/TOF MS in which samples are deposited in the vacuum onto a moving surface. High performance separation, leading to low attomole detection levels, has already been achieved. In the proposed research, the instrument will be expanded to 10 capillaries for simultaneous trace level analysis. The mass spectrometer will be operated with delayed extraction in both the linear and reflectron modes for high mass accuracy (30 ppm) and resolution (up to 10,000). Individual stained protein spots will be excised from 2-D gels and placed in a novel microfabricated device for digestion, preconcentration and desalting, followed by injection of the cleaned-up samples into the capillary array. In the protein identification mode, desalted digest samples will be concentrated by electrofocusing, followed by infusion of the mixture of matrix and sample via a liquid junction. Identification will be accomplished by peptide mapping and database searching. Protein characterization will be achieved by capillary electrophoresis (CE) separation over a 2-5 minute period using longer columns than employed for electrofocusing alone. CE separation will minimize ion suppression effects and simplify mass spectra of complex post-translationally modified peptide mixtures, as well as allowing for efficient sample utilization in future implementation of MS2 capability. As a demonstration project of the power of the proposed approach, in collaboration with Dr. George Church, proteome analysis of E. coli and S. cererisiae will be developed, for which the known genome defines all the protein that can be expressed.
