PROCESSED ANTIGEN CHARACTERIZATION BY MASS SPECTROMETRY

研究开发序列分析的仪器和方法 肽抗原呈递给免疫系统， 主要组织相容性复合体的I类和II类分子是 提出了 这是发展的必要的第一步 合成/重组疫苗或免疫系统的其它调节剂 有效对抗细菌和病毒感染，癌症， 自身免疫性疾病和组织移植排斥。 最高优先 是发展用于表征特定疾病状态的方法， 在数千个自身的复杂混合物中发现的相关抗原 缩氨酸 多级色谱法结合两种方法 微毛细管高效液相色谱 高效毛细管电泳/电喷雾串联质谱 为此目的将采用光谱测定法。 上述两个系统 将直接与细胞毒性试验相结合。 具体目标包括 （a）进一步提高串联质量的灵敏度 光谱法，以便于I类序列分析 低飞摩尔水平的肽，（B）以产生蛋白水解消化 方法，这将有助于序列分析的第二类抗原在 低飞摩尔水平，（c）对I类HLA呈递的肽进行测序， 黑色素瘤识别的人黑色素瘤细胞上的A2.1分子 特异性细胞毒性T淋巴细胞，（d）测序由 人I类分子HLA-B7、Aw 68和Aw 69，（e）以确定 肽与I类MHC分子相互作用的结构基础 并确定异种反应性HLA-A2.1识别的肽表位 限制性CTL，（f）对从HIV病毒蛋白质加工的肽进行测序 并联合呈递1类HLA-A2. 1分子，（g）至 确定内源性流感血凝素肽的范围 与由野生型小鼠产生的鼠MHC 1类Kd分子相关， 型血凝素基因和合成小基因，并鉴定内源性 结合Kd但无免疫原性的血凝素肽。

Research to develop instrumentatIon and methods for the sequence analysis of peptide antigens presented to the immune system in association with class I and class II molecules of the major histocompatibility complex is proposed. This is a necessary first step in the development of synthetic/recombinant vaccines or other modulators of the immune system that are effective against bacterial and viral infections, cancer, autoimmune disorders and tissue transplant rejection. Of highest priority is the development of methods for characterizing specific disease state associated antigens found in complex mixtures of several thousand self peptides. Multistage chromatography in conjunction with both microcapillary high performance liquid chromatography/ and high performance capillary electrophoresis/ electrospray ionization tandem mass spectrometry will be employed for this purpose. Both of the above systems will be coupled directly to cytotoxicity assays. Specific goals include the following: (a) to further improve the sensitivity of the tandem mass spectrometry method so as to facilitate sequence analysis of class I peptides at the low femtomole level, (b) to develop proteolytic digestion methods that will facilitate sequence analysis of class II antigens at the low femtomole level, (c) to sequence peptides presented by class I HLA- A2.l molecules on human melanoma cells that are recognized by melanoma specific cytotoxic T lymphocytes, (d) to sequence peptides presented by the human class I molecules, HLA-B7, Aw68, and Aw69, (e) to determine the structural basis of peptide interaction with class I, MHC molecules and to define peptide epitopes recognized by xenoreactive, HLA-A2.1 restricted CTL, (f) to sequence peptides processed from HIV virus proteins and presented in association the class 1, HLA-A2.l molecules, (g) to identify the range of endogenous influenza hemagglutinin pep tides associated with the murine MHC class 1 Kd molecule produced by the wild type hemagglutinin gene and synthetic minigenes and to identify endogenous hemagglutinin peptides that bind Kd but are not immunogenic.