This application is a request for monies that wiU be applied toward the purchase of modern MS/MS instrumentation. Thirteen projects are included in the application. The projects have been submitted by participating investigators that are located in 7 academic departments from both the main campus and the medical center. The university-wide participation demonstrates a widespread appreciation of the critical need for in-house MS/MS at Wake Forest University. AU of the investigators have used mass spectrometry in their research and the availability of MS/MS techniques wiU dramatically improve the effectiveness of these investigators. The MS/MS instrumentation will be used for the analysis of a variety of complex molecules. Samples will be ionized by one of several techniques including: electrospray, cesium ion LSIMS, or EUCI. A variety of compounds will be examined using the MS/MS instrument including small, complex organic monomers, peptides, and proteins. The VG Quattro-II triple stage quadrupole mass spectrometer having the foUowing options wiU fiU our current needs: extended mass range, EI and CI ionization source and interface, a data system controUed gas chromatograph equipped with split/splitless and cool on-column GC injectors, cesium ion LSIMS, and an API LC-MS interface for both classical and pneumaticaUy assisted low flow rate electrospray. The types of projects that will be supported by the MS/MS system fall into three broad categories. The first category of projects involves structure analysis of smaller molecules, <2,500 amu. Several projects are included from synthetic chemists desiring to determine the structures of complex organic molecules and from biochemists studying the oxidations of lipids and proteins. These investigators wiU employ LSIMS, electrospray, and El/CI techniques. The second category of projects are from investigators that need to 1) determine the masses of proteins that are then generated by site-directed mutagenesis, 2) assess modifications to proteins subjected to oxidative stress, or 3) analyze chemical modifications that are generated in mechanistic studies of enzyme action. In projects utilizing site- directed mutagenesis, it is desirable to generate proteins that have well defined modifications of the primary structure. Electrospray techniques wiU be employed to ensure that these proteins have been properly processed. In the studies on protein oxidation, the degree of oxidation and the number of oxygen atoms added to the protein will be assessed by the electrospray technique. In the mechanistic studies, reactive substrates are added to the protein and the result of the experiment is assessed by determining the change in mass of enzyme exposed to the reactive substrate. In addition to analyzing purified proteins, mixtures of peptides can be separated on the HPLC system and directly introduced into the mass spectrometer via the electrospray interface. Analysis of these fragments is essential for determining the specific region of the protein that has undergone chemical changes. The last category of projects uses MS/MS for two types of analyses. The first category will employ CID techniques to assess the fatty acid composition of phospholipids. One of the users will employ this technique to identify which phospholipids contain arachidonic acid and then assess how the distribution of arachidonic acid changes in response to the activation of metabolic pathways that utilize arachidonic acid. The second type of analysis will use CID techniques to determine the sequence of small peptides. This type of analysis will be employed to verify the site of substitution by mutagenesis, determine the primary sequence of a peptide, or determine which amino acid has been modified after free radical oxidation or after other chemical treatments. At least two significant benefits will accrue to the university from the acquisition of MS/MS instrumentation. The first benefit is an improved research environment. Eight of the thirteen investigators are junior faculty less than 40 years of age and access to MS/MS is essential for their career development. The programs of senior investigators will continue to grow by having access to advanced MS/MS techniques. The second benefit is the improved training that will be available to our graduate students and postdoctoral fellows. Page 4
