Combining Microseparations and Ion Mobility Mass Spectrometry to Probe Peptidergic Signaling in Environmental Stress

威斯康星大学麦迪逊分校的李凌军教授在化学系化学测量和成像计划的支持下，开发了多方面的分析方法，主要基于离子迁移质谱仪(IM-MS)和微分离技术，用于甲壳类动物模型系统中神经肽的大规模功能发现、定量和结构阐明。多肽在各种生理过程中起着关键作用，所选择的螃蟹模型是开发新的质谱学技术并应用这些技术获得生物相关信息的理想平台。该项目的发现有望扩大我们在神经肽家族组织和神经肽多样性在这些重要模式生物中的功能后果方面的知识库，重点放在对环境应激的反应上。在这个项目中，正在研究开发多方面离子迁移率质谱仪(IM-MS)和微分离技术来分离和分析神经肽。该项目有四个独立但相关的具体目标：(1)开发一个基于毛细管电泳联用电喷雾离子迁移率-质谱仪(ESI-IM-MS)的多维分离平台，并将其用于神经肽的分析；(2)建立离子迁移率-质谱仪(IM-MS)辅助的等压标记定量方法，并将其应用于多肽对环境胁迫的响应研究；(3)建立基于IM-MS的多肽同分异构体中D-氨基酸的快速、准确定位策略；(4)开发基于IM-MS和电子转移解离(ETD)的策略，研究气相多肽序列扰乱的机理及其对多肽从头测序的影响，并构建碰撞截面数据库以便于内源性神经肽的鉴定。在进行这个项目的过程中，一群不同的学生将接受先进的质谱学、分析分离方法和神经科学方面的培训。将继续与麦迪逊大都会学区方案合作，接待和指导高中生进行暑期研究实习，并将构建一个基于网络的多肽数据库系统。

Professor Lingjun Li of the University of Wisconsin at Madison is supported by the Chemical Measurement and Imaging Program in the Division of Chemistry to develop multi-faceted analytical methodologies, largely based on ion mobility mass spectrometry (IM-MS) and microseparation techniques for large-scale functional discovery, quantitation and structural elucidation of neuropeptides in a crustacean model system. Peptides play a key role in various physiological processes and the selected crab model is an ideal platform on which to develop novel mass spectral techniques and apply these techniques to gain biologically relevant information. The project findings are expected to expand our knowledge base in neuropeptide family organization and functional consequences of neuropeptide multiplicity in these important model organisms, with a focus on response to environmental stress. In this project, studies are being conducted to develop multi-faceted ion mobility mass spectrometry (IM-MS) and microseparation techniques for the separation and analysis of neuropeptides. The project has four separate but related specific aims: (1) to develop a multi-dimensional separation platform based on pairing capillary electrophoresis (CE) with electrospray ionization ion mobility-mass spectrometry (ESI-IM-MS), and use it for the analysis of neuropeptides; (2) to develop an ion mobility- mass spectrometry (IM-MS)-assisted isobaric tagging quantitation method and apply it to the study of peptidomic changes in response to environmental stress; (3) to develop an IM-MS-based site-specific strategy for rapid and accurate localization of D-amino acids in peptide epimers; and (4) to develop a strategy based on IM-MS and electron-transfer dissociation (ETD) to study the mechanism of gas phase peptide sequence scrambling and its impact on peptide de novo sequencing and to construct a collision cross section database to facilitate endogenous neuropeptide identification. During the course of conducting this project, a diverse group of students will be trained in advanced mass spectrometry, analytical separation methodologies and neuroscience. A partnership with the Madison Metropolitan School District program to host and mentor high school students for summer research internships will be continued and a web-based peptidome database system will be constructed.