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

在化学系化学测量和成像计划的支持下，圣母大学的梅林·布鲁宁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会对细胞和生物体中的蛋白质功能(或功能障碍)产生重大影响，即使这些蛋白质变体是由同一基因“编码”的。解决这一重要问题的标准方法是消化蛋白质(将它们分解成更小的、称为多肽的特征成分)，然后通过分离和分析得到的多肽来鉴定它们。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/或蛋白质样本复杂时。布鲁宁方法试图通过对完整蛋白质的高分辨率分离提前消化来减少这些错误识别，从而呈现与给定蛋白质相关的所有多肽以供同时分析。这种方法需要彻底但快速的消化--这是一个关键的挑战和机遇。从长远来看，得出的见解应该会加强对疟疾寄生虫等生物体产生抗药性等现象的理解。布鲁宁博士和他的学生正在通过在他们的实验室接待来自代表性不足群体的高中生，以及通过与学校教师的接触，分享对化学和生物学之间的这一努力的见解。自下而上的蛋白质组学是一种强大的技术，可以通过蛋白质分解、肽分离、串联质谱学(MS/MS)以及将质谱图与蛋白质数据库得出的预测进行比较来识别蛋白质。然而，由于这种策略通常给出的蛋白质序列覆盖率很低，可能依赖于检测特定多肽的蛋白质形式鉴定是具有挑战性的。为了解决这个问题，布鲁宁小组正在开发用毛细管区带电泳法(CZE)分离的蛋白质的在线消化。含有末端酶修饰膜或整体的毛细管正在设计，以便在电喷雾电离MS/MS分析之前进行消化。因此，来自给定蛋白质的所有蛋白水解肽的MS/MS光谱将出现在一个较窄的时间窗口中。建立时间限制标准将有助于减少假阳性和假阴性的多肽鉴定，特别是对于蛋白质形式。测试应用包括检测蛋白质混合物和组蛋白变体等模型系统中的蛋白质形式。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.