Department of Plant Systems Biology, University of Hohenheim, Stuttgart, Germany.
Curr Protoc. 2022 Jun;2(6):e425. doi: 10.1002/cpz1.425.
Proteomics and phosphoproteomics are robust tools to analyze dynamics of post-transcriptional processes during growth and development. A variety of experimental methods and workflows have been published, but most of them were developed for model plants and have not been adapted to high-throughput platforms. Here, we describe an experimental workflow for proteome and phosphoproteome studies tailored to cereal crop tissues. The workflow consists of two parallel parts that are suitable for analyzing protein/phosphoprotein from total proteins and the microsomal membrane fraction. We present phosphoproteomic data regarding quantification coverage and analytical reproducibility for example preparations from maize root and shoot, wheat leaf, and a microsomal protein preparation from maize leaf. To enable users to adjust for tissue specific requirements, we provide two different methods of protein clean-up: traditional ethanol precipitation (PC) and a recently developed technology termed single-pot, solid-phase-enhanced sample preparation (SP3). Both the PC and SP3 methods are effective in the removal of unwanted substances in total protein crude extracts. In addition, two different methods of phosphopeptide enrichment are presented: a TiO -based method and Fe(III)-NTA cartridges on a robotized platform. Although the overall number of phosphopeptides is stable across protein clean-up and phosphopeptide enrichment methods, there are differences in the preferred phosphopeptides in each enrichment method. The preferred protocol depends on laboratory capabilities and research objective. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Total protein crude extraction Basic Protocol 2: Total protein clean-up with ethanol precipitation Alternate Protocol 1: Total protein clean-up with SP3 method Basic Protocol 3: Microsomal fraction protein extraction Basic Protocol 4: Protein concentration determination by Bradford assay Basic Protocol 5: In-solution digestion with trypsin Basic Protocol 6: Phosphopeptide enrichment with TiO Alternate Protocol 2: Phosphopeptide enrichment with Fe(III)-NTA cartridges Basic Protocol 7: Peptide desalting with C material Basic Protocol 8: LC-MS/MS analysis of (phospho)peptides and spectrum matching.
蛋白质组学和磷酸化蛋白质组学是分析生长发育过程中转录后过程动态的强大工具。已经发表了多种实验方法和工作流程，但大多数方法都是针对模式植物开发的，尚未适应高通量平台。在这里，我们描述了一种针对谷物作物组织量身定制的蛋白质组和磷酸化蛋白质组研究的实验工作流程。该工作流程由两个平行部分组成，适用于分析总蛋白和微粒体膜部分的蛋白质/磷酸蛋白。我们提供了来自玉米根和茎、小麦叶和玉米叶微粒体蛋白制剂的磷酸化蛋白质组数据，以说明定量覆盖和分析重现性。为了使用户能够根据组织的具体要求进行调整，我们提供了两种不同的蛋白质清除方法：传统的乙醇沉淀（PC）和最近开发的称为单锅、固相增强样品制备（SP3）的技术。PC 和 SP3 方法在去除总蛋白粗提物中的不需要物质方面都非常有效。此外，还介绍了两种不同的磷酸肽富集方法：基于 TiO 的方法和在机器人平台上的 Fe(III)-NTA 试剂盒。尽管通过蛋白质清除和磷酸肽富集方法获得的磷酸肽总数是稳定的，但在每种富集方法中，首选的磷酸肽都存在差异。首选方案取决于实验室能力和研究目标。© 2022 作者。Wiley Periodicals LLC 出版的《当代协议》。基本方案 1：总蛋白粗提基本方案 2：用乙醇沉淀进行总蛋白清洗替代方案 1：用 SP3 方法进行总蛋白清洗基本方案 3：微粒体部分蛋白质提取基本方案 4：Bradford assay 测定蛋白质浓度基本方案 5：胰蛋白酶的溶液内消化基本方案 6：用 TiO 进行磷酸肽富集替代方案 2：用 Fe(III)-NTA 试剂盒进行磷酸肽富集基本方案 7：C 材料的肽脱盐基本方案 8：(磷酸化)肽的 LC-MS/MS 分析和谱匹配。
