SEQUENCING OF CLASS I MHC PEPTIDES BY MASS SPECTROMETRY

描述：这是对氨基酸序列的修订建议 用质谱仪分析细胞表面展示的抗原 用于识别的I类MHC(多组织相容性[原文如此])分子 由细胞毒性T淋巴细胞(CTL)或杀伤T细胞。在我们的时候 最初提交的这份提案我们已经成功地隔离了， 免疫显性H-2Kd限制性NP147-155的纯化及序列分析 从病毒转导的CT26NP细胞中分离的(核蛋白)肽和 同种异体反应性T细胞识别的TAP依赖先导肽 使用氨基肽酶、羧肽酶、 和胞外肽酶和基质辅助激光解吸电离(MALDI) 飞行时间(TOF)质谱仪。虽然这种方法使我们能够 建立高效的分离生物活性物质的层析方法 分离并建立了检测限值(细胞数量) 为了进行测序，我们感兴趣的是识别特定于 肿瘤或病毒感染(包括HIV)细胞，显示在 存在大量不完全的自身抗原； 经层析分离。因此，我们的建议是使用串联时间 使用曲线场反射器的飞行时间(TOF)质谱学 产品序列离子的同时聚焦和高性能 利用存储波形逆的四极离子陷阱质谱仪(ITMS) 傅里叶变换(SWIFT)技术用于单位质量选择、激励、 和碰撞诱导解离(CID)。 在这份修订后的提案中，我们解决了 评价者们强调了离线层析在 结合串联式仪器，并详细介绍了我们的方法 沙门氏菌特异性T细胞检测方法的建立此外，我们 在一个案例中，描述我们在减少层析工作量方面的进展 通过开发一种新的免疫沉淀策略 单次高效液相色谱分离分离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 曲线场反射质谱计，以及结果 利用SWIFT技术和脉冲重离子获得单同位素CID谱 在ITMS中进行靶向气体注入。最后，我们描述了两个新项目 目前正在进行中：相关MHC I类肽的鉴定 与MMTV感染的小鼠乳腺肿瘤细胞和天然配体识别 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.