Sanford Ethan J, Smolka Marcus B
Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.
Bio Protoc. 2021 Aug 5;11(15):e4113. doi: 10.21769/BioProtoc.4113.
Protein phosphorylation is a nearly universal signaling mechanism. To date, a number of proteomics tools have been developed to analyze phosphorylation. Phosphoproteome-wide analyses using whole cell extracts suffer from incomplete coverage, often missing phosphorylation events from low-abundance proteins. In order to increase coverage of phosphorylation sites on individual proteins of interest ("phospho-mapping"), immunoprecipitation (IP) followed by phosphoenrichment is necessary. Unfortunately, most commercially available phosphoenrichment kits are not readily scalable to the low-microgram quantities of protein present in IP eluates. Here, we describe a simple method specifically optimized for the enrichment of phosphopeptides from IP samples using an Fe-NTA based method. This method can be added downstream of any standard immunoprecipitation protocol and upstream of any MS analysis pipeline. The protocol described herein is cost effective, uses commonly available laboratory reagents, and can be used to obtain deep coverage of individual protein phosphorylation patterns, supplementary to phosphoproteomics data. Graphical abstract: Phospho-mapping workflow for a hypothetical protein of interest.
蛋白质磷酸化是一种几乎通用的信号传导机制。迄今为止，已经开发了许多蛋白质组学工具来分析磷酸化。使用全细胞提取物进行的全磷酸化蛋白质组分析存在覆盖不完整的问题，常常遗漏低丰度蛋白质的磷酸化事件。为了增加对感兴趣的单个蛋白质上磷酸化位点的覆盖（“磷酸化图谱分析”），需要进行免疫沉淀（IP）然后进行磷酸化富集。不幸的是，大多数市售的磷酸化富集试剂盒不容易扩展到IP洗脱液中存在的低微克量蛋白质。在此，我们描述了一种使用基于Fe-NTA的方法从IP样品中富集磷酸肽的简单方法，该方法经过专门优化。此方法可添加到任何标准免疫沉淀方案的下游和任何质谱分析流程的上游。本文所述的方案具有成本效益，使用常见的实验室试剂，并且可用于获得单个蛋白质磷酸化模式的深度覆盖，作为磷酸化蛋白质组学数据的补充。图形摘要：针对假设的感兴趣蛋白质的磷酸化图谱分析工作流程。
