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

本提案的目的是建立启动蛋白质化学实验室所需的仪器基础，以满足参与德克萨斯农工大学蛋白质研究的pi的需求。德克萨斯A&amp；M已经做出了每年19.5万美元的长期承诺，以聘请和留住一名杰出的博士水平的工作主任（起薪4.5万美元），以及一名高级（2.5万美元）和一名初级技术人员（1.8万美元）来支持实验室。年度预算的剩余部分将用于供应和维护（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实验室和拟议的蛋白质化学实验室将设在连续的空间内，使MALDI质谱仪可以在两个设施之间共用。生物质谱实验室的博士级工作主任，已经在校园里为100多名pi提供服务，将有助于培训蛋白质实验室的博士级主任在质谱方面利用新兴的质谱表征和测序方法。新实验室将由教务主任在各系举办研讨会，并在万维网主页上发布广告。典型蛋白质样品的分析将通过购买基于ninhydrin的氨基酸分析仪进行蛋白质和肽组成分析以及浓度测定和蛋白质测序仪来处理，该测序仪利用苯基硫代海因（PTH）化学来测序少于Ca 30残基的肽。所需的分离能力将首先通过购买配备双波长UV-Vis记录仪和毛细管区带电泳装置的可变温度HPLC来满足。这些将用于确定给定样品（经过二硫还原和变性后）中化学上不同多肽链的数量，并用于不符合要求纯度的样品的纯化。毛细管区带电泳装置尤其重要，因为它可以分离常规反相高效液相色谱无法分离的多肽。经典的SDS-PAGE /电印迹技术也将纳入蛋白质实验室，允许直接从1-或2D-SDS -PAGE凝胶进行PTH测序。最终将提供肽合成服务，但只有在A&amp；M的研究界对蛋白质测序和相关服务感到满意之后，这是我们严重不足的地方。我们的逻辑是肽合成可以与外部供应商合作完成，并与几个具有专业知识的研究实验室合作，尽管这种情况仍然不理想。到时候，凯利实验室将把他们1995年的ABI肽合成器捐赠给蛋白质化学实验室，以换取周末的特权。目前有几个小组正在使用这一仪器，它们最好在蛋白质化学实验室的主持下使用。除了处理和委派常规样品的责任（70%的工作量），实验室主任还将负责在与监督实验室的教师咨询委员会协商后将新兴技术引入实验室（15%的工作量）。我们设想质谱和蛋白质实验室的工作主管之间进行重要的互动，特别是在使非专家可以使用包络边缘方法方面。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本设施提供的技术（15%的努力）。广泛使用该设备的实验室可以选择让他们的研究生在监督下更熟悉设备的操作。由于这一政策在质谱实验室，研究生在A&amp；M了解这些技术，现在有一个walkup MAIDI仪器供他们使用。长期目标是建立一个一流的蛋白质化学实验室，以满足对蛋白质化学各个方面感兴趣的A&amp；M各院系教师的需要。这样一个设施的一个重要特点是，教师，研究生和博士后在广泛的学科变得熟悉经典和新兴的技术蛋白质表征。此外，各个部门的学生和教师最终会彼此交谈，这通常会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.