The Worcester Foundation is an independent nonprofit research institute devoted to basic research in the biological sciences. Much of the research conducted in the 22 laboratories at the Foundation deals with the functioning of proteins in cells. To provide the high quality sequence information required for the research programs at the Foundation, a protein chemistry core facility was established in 1989. The core facility is used extensively by faculty members for protein microsequencing projects, using two state-of- the-art peptide/protein sequencers. The project described in this proposal involves the acquisition of a matrix-assisted laser desorption ionization (MALDI) mass spectrometer with a time of flight (TOF) detector to be used as shared equipment in the protein chemistry core facility. Applications of mass spectrometry (MS) for protein analysis are developing rapidly and the mass spectrometer must now be considered an integral component of the modern protein chemistry lab. The Worcester Foundation core facility does not currently have a mass spectrometer. The acquisition of a MALDI-TOF mass spectrometer will complement the existing microsequencing capabilities and add important new capabilities to the protein chemistry core facility. MALDI-MS is particularly useful for projects involving biological samples because of its soft ionization technique (polypeptides are not fragmented), the ability to resolve complex mixtures, sensitivity in the femtomolar range and enhanced tolerance to sample impurities. The MALDI-TOF mass spectrometer that has been selected is the LASERMAT 2000 manufactured by Finnigan MAT Corp. The considerations affecting the selection of this particular instrument are its sensitivity, resolution, high throughput, computer capabilities and ease of operation. These characteristics are most desirable for an instrument with multiple users that will be extensively used to analyze small quantities of protein obtained from biologic al samples. Ten investigators have described research projects that can benefit immediately from the incorporation of MALDI- MS into their research programs. The number of users is expected to increase as results are communicated within the Foundation community and as new applications are developed. The projects described here cover a wide range of subjects, including the development of advanced materials, studies of protein damage and post-translational modification, the identification of regulatory proteins in cells and chemical adducts formation resulting from environmental proestrogens. Many of the projects combine mass spectrometry and peptide sequencing to identify modified peptides and provide information regarding the modification itself. Understanding the distribution of a protein between modified and unmodified forms is a critical element in elucidating signal transduction pathways and understanding the regulation of protein activities and functions in cells. The femtomolar sensitivity of the instrument permits analysis of very small quantities of cellular protein. The instrument will also be used routinely to screen samples prior to sequencing. MALDI- MS can readily establish the presence of contaminants in protein and peptide samples. In addition, proteolytic digests of newly isolated proteins can be mass mapped and compared to protein sequences in the existing databases, possibly leading to much earlier identification of the protein and reduced sequencing requirements. The instrument will also be used as an analytical tool to evaluate the integrity of recombinant proteins with a much greater resolution than the commonly employed electrophoretic systems. Plans are to develop MALDI- MS as a primary sequencing tool, as well, using new degradation chemistries. Dr. John Leszyk, the director of the protein chemistry core, will be responsible for instrument supervision and the training of new instrument users. It is an ticipated that the instrument will be used by at least half of the laboratories at the institution and that users will include approximately 8 principal investigators, 26 postdoctoral associates, 8 graduate students and 4 professional research assistants.
