AUTOMATED PROTEIN SEQUENCING BY MICRO-LC/ELECTROSPRAY MS

该项目的总体目标是开发一种高速自动化的 采用高效填充毛细管LC的蛋白质测序仪 分离、固定化酶和电喷雾质谱法。 这 系统应提供分子量和序列信息， 1-10 pmol水平的样品量和10-100 pmol水平的完整序列 pmol水平。 这种新型测序仪在技术上和经济上 与现代气相Edman测序仪竞争，但顺序 更快，更可靠，更通用，因为它 应该能够处理封闭N-末端和不寻常的氨基酸 残基 所提出的方法是基于早期的成功工作， 标准孔柱与热喷雾质谱，但需要 具有高得多的灵敏度的优点， 毛细管LC与电喷雾MS接口。 所提出的方法是氨肽酶和 具有双电荷的碰撞诱导解离的羧肽酶 胰蛋白酶肽以提供低皮摩尔量的完整序列 的胰蛋白酶片段产生和分离的在线。 在第一阶段， 建立了所提出的方法给出了可靠的，明确的序列 信息，并将表明，拟议的敏感性，现在 在商业电喷雾质量的当前性能内是可行的 质谱仪，新质谱技术的进一步改进 适用于蛋白质和肽被认为是可能的， MS的灵敏度提高了，这种新方法的灵敏度也将提高 to sequencing测序.

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.