Acquisition of a Multiple Peptide Synthesizer and Laser Desorption Mass Spectrometer

耶鲁大学的W.M.凯克基金会生物技术资源实验室为耶鲁大学、康奈尔大学、哥伦比亚大学、哈佛医学院、约翰霍普金斯医学院、西奈山医学院、洛克菲勒大学、斯隆-凯特林癌症中心、德克萨斯农工大学、加利福尼亚大学、伊利诺斯大学、密歇根大学、华盛顿大学和华盛顿大学的近600名主要研究人员提供全方位的蛋白质和核酸分析和合成服务。德克萨斯州和弗吉尼亚州以及另外123所院校。该设施每年完成10,000多项服务，包括约750种蛋白质和肽的测序。后一种样品绝大多数是hplc纯化的多肽，这些多肽要么是从MHC分子中洗脱出来的，要么是从Keck设施对SDS PAGE分离的蛋白质进行的胰蛋白酶消化中分离出来的。尽管所有这些肽在HPLC上都有对称的吸光峰，但氨基酸测序表明，事实上，近三分之一的样品要么是肽的混合物，要么是由考马斯蓝和其他试剂产生的伪峰，要么是在测序前由于吸附在塑料表面而丢失的。所要求的激光解吸质谱仪（LDMS）能够常规测定50- 500飞莫量的肽的质量，在大多数情况下，能够预先鉴定那些在预期质量范围内仅含有单一物种的三分之二的样品（对于10-40残基肽），因此适合进行氨基酸测序。除了防止对不合适的样品进行不必要的测序外，所得到的质量将确认仅通过Edman降解暂时分配的残基，并将确定这些肽的长度，这在表征MHC结合肽时特别值得关注。LDMS的其他应用是描述蛋白质的翻译后修饰，并可能作为寡核苷酸合成的质量控制监测器。另一个需要的仪器是一台能够同时合成和自动切割96个多肽的多肽合成器，这将需要大量价格经济、质量高的合成多肽。目前，耶鲁大学的几个实验室正在使用人工多肽合成系统每年合成多达100个多肽。显然，获得多肽合成器将大大提高这些实验室的研究效率，并将使这些基本低聚物的成本在更多研究人员的负担范围内。如果资金提供，这两种仪器将立即被用于大量重要的研究项目，范围从描述T细胞反应中与MHC分子结合的肽的特征到增加我们对自身免疫的生化基础的理解，蛋白质折叠，甲状旁腺样激素相关蛋白的翻译后加工，活化B淋巴细胞中的同型开关重组到肽核酸的生物学应用。凯克设施的高水平技术专长实际上保证了这些仪器将得到专业的维护，该设施为耶鲁大学和非耶鲁大学的大量研究人员提供服务，确保了这些设备对生化研究的积极影响将是巨大的，并将远远超出耶鲁大学。

The W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University provides a full range of protein and nucleic acid analytical and synthetic services to nearly 600 principal investigators at Yale University, Cornell University, Columbia University, Harvard Medical School, Johns Hopkins Medical School, Mt. Sinai School of Medicine, Rockefeller University, Sloan-Kettering Cancer Center, Texas A&M University, and the universities of California, Illinois, Michigan, Texas and Virginia as well as at another 123 institutions. Each year this facility completes more than 10,000 services including the sequencing of ~750 proteins and peptides. The vast majority of the latter samples are HPLC-purified peptides that have either been eluted from MHC molecules or that have been isolated from tryptic digests carried out by the Keck Facility on SDS PAGE- separated proteins. Although all these peptides gave symmetrical absorbance peaks upon HPLC, amino acid sequencing demonstrated that nearly one-third of these samples were, in fact, either mixtures of peptides, artifact peaks resulting from Coomassie Blue and other reagents or, were lost prior to sequencing due to adsorption onto plastic surfaces. The ability of the requested laser desorption mass spectrometer (LDMS) to routinely determine masses on 50- 500 femtomole amounts of peptide will, in most instances, enable the prior identification of those two-thirds of the samples which contain only a single species in the expected mass range (for a 10-40 residue peptide) and which are therefore suitable for amino acid sequencing. In addition to preventing the needless sequencing of unsuitable samples, the resulting masses will confirm residues which would have been only tentatively assigned by Edman degradation and will establish the length of these peptides, which is of particular concern in characterizing MHC bound peptides. Other applications envisioned for LDMS are in characterizing posttranslational modificati ons of proteins and, possibly, as a quality control monitor of oligonucleotide synthesis. The other instrument that is requested, a multiple peptide synthesizer capable of the simultaneous synthesis and automated cleavage of 96 peptides, will need the increasing demand for large numbers of economically priced, high quality synthetic peptides. Currently, several laboratories at Yale are synthesizing as many as 100 peptides/year using a manual system of multiple peptide synthesis. Obviously, acquisition of a multiple peptide synthesizer would significantly increase research productivity in these laboratories and would bring the cost of these essential oligomers within reach of many more investigators. If funds are provided, these two instruments will immediately be brought to bear on a large number of important research projects that range from characterizing peptides bound to MHC molecules in T cell responses to increasing our understanding of the biochemical basis of autoimmunity, protein folding, posttranslational processing of a parathyroid-like hormone related protein, isotype switch recombination in activated B lymphocytes to biological applications of peptide nucleic acids. The high level of technical expertise in the Keck Facility virtually guarantees that these instruments would be expertly maintained and the extremely large number of Yale and non- Yale investigators served by this facility ensures that the positive impact of this equipment on biochemical research would be large and would extend well beyond Yale University.