SEQUENCING OF CLASS I MHC PEPTIDES BY MASS SPECTROMETRY

描述：这是氨基酸序列的修订建议 通过质谱分析展示在细胞表面上的抗原， 用于识别的I类MHC（多种组织相容性[Sic ]）分子 细胞毒性T淋巴细胞（CTL）或杀伤T细胞。 当时， 我们成功地分离出了最初提交的这个提案， 纯化并测序免疫显性H-2Kd限制性NP 147 -155 从病毒转导的CT 26 NP细胞分离的（核蛋白）肽和一种 特异性同种异体反应性T细胞识别的接头依赖性前导肽 使用氨基肽酶，羧肽酶， 和外肽酶和基质辅助激光解吸/电离（MALDI） 飞行时间（TOF）质谱法。 虽然这种方法使我们能够 开发高效的色谱分离方法， 馏分，并已建立检测限（细胞数） 测序所需的，我们的兴趣是确定抗原特异性， 肿瘤或病毒感染（包括HIV）的细胞，显示在 存在大量不能完全的自身抗原;y 色谱分离。 因此，我们的建议是采用串联时间 飞行时间（TOF）质谱，使用曲场反射器， 产品序列离子的同时聚焦和高性能 使用存储波形反转的四极离子阱质谱仪 傅立叶变换（SWIFT）技术用于单位质量选择、激励 碰撞诱导解离（CID） 在这一修订提案中，我们处理了 评论家，强调离线色谱在 与串联仪器相结合，并详细介绍了我们的方法， 沙门氏菌特异性T细胞测定的开发。 另外我们 描述我们在减少色谱工作方面的进展，在一个案例中， 通过开发一种新的免疫沉淀策略， 使用单一HPLC分级分离IB类抗原。 我们也 描述我们最近在性能（和商业化）方面的改进， 弯曲场反射质谱仪，以及结果， 使用SWIFT技术获得单同位素CID谱，并使用脉冲重 ITMS中的目标气体注入。 最后，我们描述了两个新的项目， 目前正在进行中：鉴定MHC I类肽相关 用MMTV感染的小鼠乳腺肿瘤细胞， 在胸腺的T细胞选择过程中。

DESCRIPTION: This is a revised proposal for the amino acid sequence analysis by mass spectrometry of antigens displayed on cell surfaces by Class I MHC (multiple histocompatibility [Sic ]) molecules for recognition by cytotoxic T lymphocytes (CTL) or killer T cells. At the time of our original submission of this proposal we had been successful in isolating, purifying, and sequencing an immunodominant H-2Kd-restricted NP147-155 (nucleoprotein) peptide isolated from virally transduced CT26NP cells and a Tap-dependent leader peptide recognized by alloreactive T cells specific for Class IB antigen using combinations of aminopeptidases, carboxypeptidases, and exopeptidases and matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. While this approach has enabled us to develop efficient chromatographic methods for isolation of bioactive fractions and has established the detection limits (number of cells) required for sequencing, our interest is in identifying antigens specific to tumor or virally infected (including HIV) cells, that are displayed int the presence of a large number of self antigens that cannot be complete;y isolated chromatographically. Thus, our proposal was to employ tandem time of flight (TOF) mass spectrometry using a curved-field reflectron for simultaneous focussing of product sequence ions and a high performance quadrupole ion trap mass spectrometer (ITMS) using stored waveform inverse Fourier transform (SWIFT) techniques for unit mass selection, excitation, and collision induced dissociation (CID). In this revised proposal we address several concerns expressed by the reviewers, stressing the continued importance of off-line chromatography in combination with tandem instruments and detailing our methods for the development of a Salmonella -specific T cell assay. In addition, we describe our progress in reducing the chromatographic effort, in one case through development of a novel immunoprecipitation strategy for the isolation of Class IB antigen using a single HPLC fractionation. We also describe our recent improvements in performance (and commercialization) of the curved field reflectron mass spectrometer, as well as results in obtaining monoisotopic CID spectra using SWIFT techniques and pulsed heavy target gas injection in an ITMS. Finally, we describe two new projects that are now underway: the identification of MHC class I peptides associated with MMTV-infected mouse mammary tumor cells, and natural ligands recognized during T-cell selection in the thymus.