A Rapid Screening Assay to Search for Phosphorylated Proteins in Tissue Extracts

Reversible protein phosphorylation is an essential mechanism in the regulation of diverse biological processes, nonetheless is frequently altered in disease. As most phosphoproteome studies are based on optimized in-vitro cell culture studies new methods are in need to improve de novo identification and characterization of phosphoproteins in extracts from tissues. Here, we describe a rapid and reliable method for the detection of phosphoproteins in tissue extract based on an experimental strategy that employs 1D and 2D SDS PAGE, Western immunoblotting of phosphoproteins, in-gel protease digestion and enrichment of phosphorpeptides using metal oxide affinity chromatography (MOAC). Subsequently, phosphoproteins are identified by MALDI-TOF-MS/MS with the CHCA-TL or DHB ML sample matrix preparation method and further characterized by various bioinformatic software tools to search for candidate kinases and phosphorylation-dependent binding motifs. The method was applied to mouse lung tissue extracts and resulted in an identification of 160 unique phosphoproteins. Notably, TiO2 enrichment of pulmonary protein extracts resulted in an identification of additional 17 phosphoproteins and 20 phosphorylation sites. By use of MOAC, new phosphorylation sites were identified as evidenced for the advanced glycosylation end product-specific receptor. So far this protein was unknown to be phosphorylated in lung tissue of mice. Overall the developed methodology allowed efficient and rapid screening of phosphorylated proteins and can be employed as a general experimental strategy for an identification of phosphoproteins in tissue extracts.

可逆蛋白质磷酸化是调控多种生物学过程的核心机制，却常于疾病状态下发生异常改变。由于绝大多数磷酸化蛋白质组学（phosphoproteome）研究均基于优化的体外细胞培养模型，当前亟需开发新方法，以实现组织提取物中磷酸化蛋白质的从头鉴定与表征。本文报道了一种快速可靠的组织提取物磷酸化蛋白质检测方法，该实验策略整合了一维及二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS PAGE）、磷酸化蛋白质免疫印迹、凝胶内蛋白酶酶解，以及采用金属氧化物亲和色谱（MOAC）富集磷酸肽的步骤。随后，采用CHCA-TL或DHB ML基质样品制备法结合基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF-MS/MS）对磷酸化蛋白质进行鉴定，并通过多种生物信息学软件工具进一步分析，以筛选候选激酶及磷酸化依赖的结合基序。该方法应用于小鼠肺组织提取物后，成功鉴定出160种特有磷酸化蛋白质。值得注意的是，对肺组织提取物进行二氧化钛（TiO2）富集后，额外鉴定出17种磷酸化蛋白质及20个磷酸化位点。通过金属氧化物亲和色谱技术，研究者还鉴定到新的磷酸化位点，例如晚期糖基化终产物特异性受体（advanced glycosylation end product-specific receptor）——此前该蛋白在小鼠肺组织中尚未被报道存在磷酸化修饰。总体而言，本研究所开发的方法学可实现磷酸化蛋白质的高效快速筛选，可作为通用实验策略用于组织提取物中磷酸化蛋白质的鉴定工作。