Acquisition of Instrumentation for the Development of a Protein Characterization Laboratory to Serve the Needs of the Texas A&M Scientific Community

这项建议的目的是建立启动蛋白质化学实验室所需的仪器基础，以满足德克萨斯农工大学参与蛋白质研究的PI的需求。德克萨斯A&A&M已经做出了每年19.5万美元的长期承诺，将聘用和保留一名杰出的博士级别的工作主任(45K美元Start)以及一名高级(25K)和初级技术员(18K)来支持实验室。年度预算的其余部分将用于用品和维护(10.7万美元)。化学系提供了足够的空间，以便已建立的生物质谱学实验室和拟议的蛋白质化学实验室将设在毗连的空间内，以便在设施之间共享一台MALDI质谱仪。生物质谱学实验室的博士工作主任已经在校园内为100多个PI提供服务，他将帮助培训蛋白质实验室的博士级别的质谱学主任，使其能够利用新兴的质谱学表征和测序方法。新实验室将通过各系的研讨会、教研室主任和万维网上的主页进行宣传。对典型蛋白质样本的分析将通过购买一台用于蛋白质和多肽组成分析以及浓度测定的吲哚三酮氨基酸分析仪和一台蛋白质测序仪来处理，该测序仪利用苯硫代海因(PTH)化学对低于钙30个残基的多肽进行排序。所需的分离能力将首先通过购买配备双波长UV-Vis记录器和毛细管区带电泳仪的可变温度高效液相色谱仪来满足。它们将用于确定给定样品(在二硫键还原和变性后)中化学上不同的多肽链的数量，并用于提纯不具有所需纯度的样品。毛细管区带电泳仪的建立尤其重要，因为它可以分离常规反相高效液相色谱分离不能分离的多肽。经典的十二烷基硫酸钠-PAGE/电印迹技术也将被纳入蛋白质实验室，以允许直接从1-或2D-十二烷基硫酸钠-PAGE凝胶中进行PTH测序。最终将提供多肽合成服务，但只有在A&M的研究社区对蛋白质测序和相关服务感到满意之后，我们才会提供这方面的服务，这是我们严重不足的地方。我们的逻辑是，多肽合成可以与外部供应商合作，并与几个拥有专业知识的研究实验室合作完成，尽管这种情况仍然不理想。到时候，凯利实验室将把他们1995年生产的ABI多肽合成器捐赠给蛋白质化学实验室，以换取周末的特权。该仪器目前正被几个小组使用，它们的使用最好是在蛋白质化学实验室的主持下进行。除了处理和委派常规样品的责任(70%的努力)外，实验室主任还将负责在咨询负责监督实验室的教员咨询委员会(15%的努力)后，将新兴技术引入实验室。我们预计质谱学和蛋白质实验室的工作主管之间会有重大的互动，特别是在向非专家提供包络边缘方法方面。主任还将与教师咨询委员会合作，负责定期宣传和教授迷你课程，以培训本科生、研究生和博士后如何最好地利用该设施提供的技术(15%的努力)。广泛使用该设施的实验室可以选择让他们的研究生以一种监督的方式更密切地熟悉设备的操作。由于质谱学实验室的这一政策，A&Amm的研究生对这些技术了如指掌，现在有了一台无人值守的Maidi仪器供他们使用。我们的长期目标是建立一个一流的蛋白质化学实验室，满足对蛋白质化学各个方面感兴趣的不同部门的A&M教职员工的需求。这种设施的一个重要特征是，广泛学科的教师、研究生和博士后对蛋白质表征的经典和新兴技术非常熟悉。此外，不同系的学生和教职员工相互交谈，经常导致研究小组之间的实质性合作。

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.