Functional Genomics Section and Cell Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland.
Mass Spectrometry Facility, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland.
Curr Protoc. 2024 Apr;4(4):e1028. doi: 10.1002/cpz1.1028.
Proteomics and phosphoproteomics play crucial roles in elucidating the dynamics of post-transcriptional processes. While experimental methods and workflows have been established in this field, a persistent challenge arises when dealing with small samples containing a limited amount of protein. This limitation can significantly impact the recovery of peptides and phosphopeptides. In response to this challenge, we have developed a comprehensive experimental workflow tailored specifically for small-scale samples, with a special emphasis on neuronal tissues like the trigeminal ganglion. Our proposed workflow consists of seven steps aimed at optimizing the preparation of limited tissue samples for both proteomic and phosphoproteomic analyses. One noteworthy innovation in our approach involves the utilization of a dual enrichment strategy for phosphopeptides. Initially, we employ Fe-NTA Magnetic beads, renowned for their specificity and effectiveness in capturing phosphopeptides. Subsequently, we complement this approach with the TiO-based method, which offers a broader spectrum of phosphopeptide recovery. This innovative workflow not only overcomes the challenges posed by limited sample sizes but also establishes a new benchmark for precision and efficiency in proteomic investigations. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Protein extraction and digestion Basic Protocol 2: TMT labeling and peptide cleanup Basic Protocol 3: IMAC Fe-NTA magnetic beads phosphopeptide enrichment Basic Protocol 4: TiO2 enrichment Basic Protocol 5: Fe-NTA phosphopeptide Enrichment Basic Protocol 6: High pH peptide fractionation Basic protocol 7: LC-MS/MS analysis and database search.
蛋白质组学和磷酸化蛋白质组学在阐明转录后过程的动态方面发挥着关键作用。虽然在该领域已经建立了实验方法和工作流程，但当处理含有有限量蛋白质的小样本时，仍然存在一个持续的挑战。这种限制会严重影响肽和磷酸肽的回收。针对这一挑战，我们开发了一种专门针对小样本的综合实验工作流程，特别强调三叉神经节等神经元组织。我们提出的工作流程包括七个步骤，旨在优化有限组织样本的制备，以进行蛋白质组学和磷酸化蛋白质组学分析。我们方法中的一个创新之处是使用双富集策略来富集磷酸肽。最初，我们使用 Fe-NTA 磁珠，该磁珠因其特异性和捕获磷酸肽的有效性而闻名。随后，我们用 TiO2 方法补充这种方法，该方法提供了更广泛的磷酸肽回收范围。这种创新的工作流程不仅克服了样本量有限带来的挑战，而且为蛋白质组学研究的精度和效率确立了新的基准。2024 年出版。本文是美国政府的一项工作，在美国属于公有领域。Wiley Periodicals LLC 出版的《当代协议》。基础方案 1：蛋白质提取和消化基础方案 2：TMT 标记和肽纯化基础方案 3：IMAC Fe-NTA 磁珠磷酸肽富集基础方案 4：TiO2 富集基础方案 5：Fe-NTA 磷酸肽富集基础方案 6：高 pH 肽分级基础方案 7：LC-MS/MS 分析和数据库搜索。
