Acquisition of a Triple Stage Quadrupole Mass Spectrometer

本申请是一项资金申请，用于购买现代MS/MS仪器。申请中包括13个项目。这些项目是由来自主校区和医学中心的7个学术部门的参与研究人员提交的。整个大学的参与表明，维克森林大学对内部MS/MS的迫切需求得到了广泛的认识。所有的研究人员都在他们的研究中使用了质谱学，MS/MS技术的可用性极大地提高了这些研究人员的效率。MS/MS仪器将用于各种复杂分子的分析。样品将通过几种技术中的一种进行电离，包括：电喷雾、铯离子LSIMS或EUCI。将使用MS/MS仪器检测各种化合物，包括小而复杂的有机单体、多肽和蛋白质。VG Quattro-II三级四极质谱计具有以下选项以满足我们当前的需求：更宽的质量范围、EI和CI电离源和接口、配备分离式/无分离式和冷柱上GC进样器的数据系统控制的气相色谱仪、铯离子LSIMS以及用于经典和气动辅助低流速电喷雾的API LC-MS接口。MS/MS系统将支持的项目类型分为三大类。第一类项目涉及2500 amu小分子的结构分析。有几个项目包括希望确定复杂有机分子结构的合成化学家和研究脂类和蛋白质氧化的生物化学家。这些研究人员使用LSIMS、电喷雾和EL/CI技术。第二类项目来自研究人员，他们需要1)确定通过定点突变产生的蛋白质的质量，2)评估对遭受氧化应激的蛋白质的修饰，或3)分析在酶作用的机制研究中产生的化学修饰。在利用定点突变的项目中，希望产生的蛋白质具有明确的一级结构修饰。电喷雾技术将被用来确保这些蛋白质得到适当的加工。在蛋白质氧化的研究中，将通过电喷雾技术来评估蛋白质的氧化程度和添加到蛋白质中的氧原子的数量。在机理研究中，将反应底物添加到蛋白质中，并通过测定暴露在反应底物中的酶的质量变化来评估实验结果。除了分析纯化的蛋白质外，还可以在高效液相系统上分离多肽混合物，并通过电喷雾界面将其直接引入质谱仪。对这些片段的分析对于确定经历了化学变化的蛋白质的特定区域至关重要。最后一类项目使用MS/MS进行两种类型的分析。第一类将使用CID技术来评估磷脂的脂肪酸组成。其中一位使用者将使用这项技术来识别哪些磷脂含有花生四烯酸，然后评估花生四烯酸的分布如何随着利用花生四烯酸的代谢途径的激活而变化。第二类分析将使用CID技术来确定小肽的序列。这种类型的分析将用于验证突变取代的位置，确定多肽的初级序列，或确定在自由基氧化或其他化学处理后哪些氨基酸被修饰。购买MS/MS仪器至少会给大学带来两个显著的好处。第一个好处是改善了研究环境。13名研究人员中有8名是40岁以下的初级教员，获得MS/MS对他们的职业发展至关重要。通过获得先进的MS/MS技术，高级调查人员的项目将继续增长。第二个好处是我们的研究生和博士后研究员将获得更好的培训。第4页

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