The purpose of this proposal is to establish the instrumentation base required for initiating a protein chemistry laboratory to serve the needs of PIs involved in protein research at Texas A&M University. Texas A&M has made a long term commitment of $195,000/year to hire and retain an exceptional Ph.D. level working director ($45K start ) as well as a senior (25K) and junior technician (18K) to support the laboratory. The remainder of the annual budget will be for supplies and maintenance ($107K). Sufficient space has been provided in the department of chemistry so that the established Laboratory for Biological Mass Spectrometry and the proposed Protein Chemistry Laboratory will located in contiguous space allowing a MALDI mass spectrometer to be shared between the facilities. The Ph.D. working director of the biological mass spectrometry laboratory, which is already serving over 100 PIs on campus, will be instrumental in training the Ph.D. level director of the Protein laboratory in mass spectrometry to take advantages of the emerging mass spectrometric characterization and sequencing methods. The new laboratory will be advertised by seminars in various departments by the faculty director and by a home page on the world wide web. The analysis of typical protein samples will be handled by purchasing a ninhydrin-based Amino acid analyzer for protein and peptide composition analysis as well as for concentration determinations and a protein sequencer which utilizes phenylthiohydantoin (PTH) chemistry for sequencing peptides of less than Ca thirty residues. The required separations capabilities will be met initially by the purchase of a variable temperature HPLC fitted with a dual wavelength UV-Vis recorder and a capillary zone electrophoresis set up. These will be used for determining the number of chemically different polypeptide chains in a given sample (after disulfide reduction and denaturation) and for purification of samples which are not of the required purity. The capillary zone electrophoresis set up is particularly important as it can separate polypeptides that cannot be separated by routine RP-HPLC. Classical SDS-PAGE / electroblotting technology will also be incorporated into the Protein laboratory to allow PTH sequencing directly from a 1- or 2D-SDS PAGE gel. Eventually peptide synthesis services will be offered, but only after the research community at A&M is pleased with protein sequencing and related services which is where we are severely deficient. Our logic here is that peptide synthesis can be accomplished with outside vendors and in collaboration with several research laboratories with expertise, although this situation is still not ideal. When the time comes the Kelly Laboratory will donate their 1995 vintage ABI peptide synthesizer to the Protein Chemistry laboratory in return for weekend privileges. This instrument is currently being utilized by several groups whose use would be best accommodated under the auspices of the laboratory for protein chemistry. In addition to handling and delegating responsibilities for routine samples (70% effort) the director of the laboratory will be responsible for bringing emerging technology into the laboratory after consulting with the faculty advisory committee which oversees the laboratory (15% effort). We envision significant interaction between the working directors of the mass spectrometry and protein laboratories, particularly with regard to making edge of the envelope methods available to non-experts. The director in collaboration with the faculty advisory committee would also be responsible for advertising and teaching mini-courses periodically to train undergraduate, graduate students and post-doctorals how to best use the technology available in this facility(l5% effort). Laboratories who make extensive use of the facility may elect to have their graduate students become more intimately familiar with the opera tion of the equipment m a supervised manner. As a result of this policy in the mass spectrometry laboratory the graduate students at A&M understand these techniques well enough that there now is a walkup MAIDI instrument for their use. The long term objective is to build a superb protein chemistry laboratory which serves the needs of A&M faculty in a variety of departments interested in various aspects of protein chemistry. An important feature of such a facility is that the faculty, graduate students and post-doctorals in a wide range of disciplines become intimately familiar with classical and emerging technology for protein characterization. In addition students and faculty in various departments end up talking to one another often leading to substantive collaborations between research groups.
