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Membrane and Monolith Enzyme Reactors for Proteolysis After Capillary Electrophoresis to Enhance Detection of Proteoforms

用于毛细管电泳后蛋白水解的膜和整体式酶反应器，以增强蛋白质形式的检测

基本信息

批准号：
1903967
负责人：
Merlin Bruening
金额：
$ 37.5万
依托单位：
University of Notre Dame
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1903967&HistoricalAwards=false
关键词：
Membrane Monolith Enzyme Reactors Proteolysis

项目摘要

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Merlin Bruening and his group at the University of Notre Dame are working to develop a new method to characterize small changes in the composition of proteins - key elements in the chemistry of life. Subtle differences in composition can have large impacts on protein function (or malfunction) in cells and organisms, even when the protein variants are "coded" by the same gene. A standard approach to this important problem is to digest proteins (break them into smaller, characteristic constituents known as peptides) and then identify them by separation and analysis of the resulting peptides. This can lead to misidentifications (false positives and false negatives), especially when the structural differences are minor and/or the protein sample is complex. The Bruening approach seeks to reduce these misidentifications by preceding digestion with a high-resolution separation of the intact proteins, thereby presenting for simultaneous analysis all the peptides associated with a given protein. The approach requires thorough but rapid digestion - a key challenge and opportunity. In the long run, the insights derived should enhance understanding of phenomena like the development of drug resistance by organisms such as the malaria parasite. Dr. Bruening and his students are sharing insights about this effort at the interface between chemistry and biology through hosting high school students from underrepresented groups in their lab, and through outreach to school teachers.Bottom-up proteomics is a powerful technique for identifying proteins via proteolysis, peptide separation, tandem mass spectrometry (MS/MS), and comparison of mass spectra to the predictions derived from a protein database. However, because this strategy often gives low protein sequence coverage, proteoform identification, which may depend on detecting a specific peptide, is challenging. To address this problem, the Bruening group is developing in-line digestion of proteins separated using capillary zone electrophoresis (CZE). Capillaries containing terminal enzyme-modified membranes or monoliths are being designed to enable digestion just prior to electrospray ionization MS/MS analysis. Thus, the MS/MS spectra of all the proteolytic peptides from a given protein will appear in a narrow time window. Creation of a time-bin criterion will help to decrease both false-positive and false-negative peptide identifications, particularly for proteoforms. Test applications include detection of proteoforms in model systems such as protein mixtures and histone variants.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在化学系化学测量和成像项目的支持下，圣母大学的Merlin Bruening教授和他的团队正在努力开发一种新方法来表征蛋白质组成的微小变化-生命化学中的关键元素。组成上的细微差异可能对细胞和生物体中的蛋白质功能（或故障）产生巨大影响，即使蛋白质变体由相同的基因“编码”。解决这一重要问题的标准方法是消化蛋白质（将其分解为较小的特征成分，称为肽），然后通过分离和分析所得肽来识别它们。这可能导致错误识别（假阳性和假阴性），特别是当结构差异很小和/或蛋白质样品很复杂时。 Bruening方法试图通过在消化之前对完整蛋白质进行高分辨率分离来减少这些错误识别，从而同时分析与给定蛋白质相关的所有肽。这种方法需要彻底而迅速的消化--这是一个关键的挑战和机遇。从长远来看，所得出的见解应该会增强对诸如疟疾寄生虫等生物体产生抗药性等现象的理解。 Bruening博士和他的学生通过在实验室接待来自代表性不足群体的高中生，并通过与学校教师的联系，在化学和生物学之间的界面上分享关于这一努力的见解。自下而上的蛋白质组学是一种强大的技术，通过蛋白质水解，肽分离，串联质谱（MS/MS）以及质谱与蛋白质数据库预测的比较来鉴定蛋白质。然而，由于这种策略通常给出低蛋白质序列覆盖率，可能依赖于检测特定肽的蛋白质型鉴定是具有挑战性的。为了解决这个问题，Bruening小组正在开发使用毛细管区带电泳（CZE）分离的蛋白质的在线消化。正在设计含有末端酶修饰的膜或整料的封端剂，以使消化恰好在电喷雾电离MS/MS分析之前进行。因此，来自给定蛋白质的所有蛋白水解肽的MS/MS谱将出现在窄的时间窗口中。建立一个时间箱标准将有助于减少假阳性和假阴性肽鉴定，特别是蛋白质型。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。