AUTOMATED PROTEIN SEQUENCING BY MICRO-LC/ELECTROSPRAY MS

通过 MICRO-LC/ELECTROSPRAY MS 进行自动蛋白质测序

• 批准号: 3498648
• 负责人: MARVIN L VESTAL
• 金额: $ 5万
• 依托单位: VESTEC CORPORATION
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1991-12-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3498648
• 关键词: aminopeptidase; bioengineering /biomedical engineering; biomedical automation; biomedical equipment development; carboxypeptidase; mass spectrometry

The overall goal of this project is to develop a high-speed automated protein sequencer employing high-performance packed capillary LC separation, immobilized enzymes, and electrospray mass spectrometry. This system should provide molecular weight and sequence information using sample amounts in the 1-10 pmol level and complete sequences at the 10-100 pmol level. This new sequencer should be technically and economically competitive with the modern gas phase Edman sequencer, but orders of magnitude faster, and somewhat more reliable and versatile in that it should be able to deal with blocked N-termini and unusual amino acid residues. The proposed approach is based on earlier successful work using standard-bore columns with thermospray mass spectrometry, but takes advantage of the much higher sensitivities which are possible with packed capillary LC interfaced to electrospray MS. An important new component to the proposed approach is the combination of both aminopeptidases and carboxypeptidases with collision induced dissociation of doubly charged tryptic peptides to provide complete sequences on low picomole quantities of tryptic fragments produced and isolated on-line. In phase I it will be established that the proposed approach gives reliable, unambiguous sequence information, and it will be shown that the proposed sensitivities are now feasible within the present performance of commercial electrospray mass spectrometers, further improvement in new mass spectrometric techniques applicable to proteins and peptides are considered likely, and as the sensitivity of the MS improves so will the sensitivity of this new approach to sequencing.

该项目的总体目标是开发高速自动化 使用高性能毛细管LC的蛋白质测序仪 分离，固定酶和电喷雾质谱法。 这 系统应使用 1-10 PMOL水平的样品量和10-100的完整序列 PMOL水平。 这个新的音序器在技术上和经济上应该是 与现代气相Edman Sequencer竞争，但 幅度更快，更可靠和多才多艺 应该能够处理阻塞的N末端和异常氨基酸 残留物。 拟议的方法是基于早期成功的工作 带有热刺质谱的标准孔柱，但采用 包装的更高敏感性的优势 毛细管LC连接到电喷雾MS。 一个重要的新组成部分 提出的方法是两种氨基肽酶和 羧肽酶与碰撞引起的双重电荷解离 胰蛋白酶肽可在低丘疹量上提供完整的序列 在线产生和孤立的胰蛋白酶片段。 在第一阶段将是 确定所提出的方法给出了可靠，明确的序列 信息，将显示提出的敏感性现在是 在目前的商业电喷雾质量的性能中可行 光谱仪，新的质谱技术进一步改进 适用于蛋白质和肽被认为可能是可能的，作为 MS的敏感性提高了，这种新方法的敏感性也会提高 进行排序。