Acquisition of a Multiple Peptide Synthesizer and Laser Desorption Mass Spectrometer

W. M.耶鲁大学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、麻省理工学院等近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。西奈医学院，洛克菲勒大学，斯隆-凯特琳癌症中心，得克萨斯州A M大学，以及加州，伊利诺伊州，密歇根州，得克萨斯州和弗吉尼亚州的大学以及另外123个机构。 该设施每年完成超过10，000项服务，包括约750种蛋白质和肽的测序。 后者样品中的绝大多数是HPLC纯化的肽，其已经从MHC分子洗脱或已经从胰蛋白酶解物分离，所述胰蛋白酶解物由Keck Facility对SDS PAGE分离的蛋白进行。 尽管所有这些肽在HPLC上产生对称的吸收峰，但氨基酸测序证明，这些样品中的近三分之一实际上是肽的混合物、考马斯蓝和其他试剂产生的伪峰，或者在测序前由于吸附到塑料表面上而丢失。 在大多数情况下，所要求的激光解吸质谱仪（LDMS）常规测定50- 500飞摩尔量的肽质量的能力将能够预先鉴定仅含有预期质量范围内的单一物质（对于10-40个残基的肽）并因此适用于氨基酸测序的三分之二样品。 除了防止对不合适的样品进行不必要的测序之外，所得的质量将确认仅通过Edman降解暂时指定的残基，并将确定这些肽的长度，这在表征MHC结合肽中特别重要。 LDMS的其他应用是表征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。 所要求的另一种仪器，即能够同时合成和自动切割96种肽的多肽合成仪，将需要对大量经济价格的高质量合成肽的日益增长的需求。 目前，耶鲁大学的几个实验室正在使用多肽合成的手动系统合成多达100肽/年。 显然，获得多肽合成仪将显著提高这些实验室的研究生产力，并将使这些基本低聚物的成本在更多研究人员的能力范围内。 如果获得资金，这两种仪器将立即用于大量重要的研究项目，从表征T细胞反应中与MHC分子结合的肽，到增加我们对自身免疫，蛋白质折叠，甲状旁腺样激素相关蛋白的翻译后加工的生化基础的理解，从活化B淋巴细胞中同种型转换重组到肽核酸的生物学应用。 高水平的技术专业知识在凯克设施几乎保证了这些仪器将得到专业维护和耶鲁大学和非耶鲁大学的研究人员由这个设施服务的数量非常大，确保了积极的影响，这种设备的生化研究将是巨大的，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.