Characterization of the phosphoproteome in human bronchoalveolar lavage fluid.
Characterization of the phosphoproteome in human bronchoalveolar lavage fluid.
复制标题
DOI：
10.1155/2012/460261
复制
发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Beranova-Giorgianni S
中科院分区：
文献类型：
--
作者：
Giorgianni F;Mileo V;Desiderio DM;Catinella S;Beranova-Giorgianni S
通过磷蛋白质组学方法对人体体液中蛋白质磷酸化进行全球范围的检测是一个新兴的研究领域，有可能对发现新的生物标志物做出重大贡献。在这项试点工作中，我们分析了来自非疾病受试者的人支气管肺泡灌洗液(BAL)中的磷蛋白质组。主要目的是评估在人类BAL中探测磷酸化蛋白的可行性，并获得BAL磷酸化蛋白的初步目录，包括蛋白质鉴定和对其磷酸化位点的准确描述。我们使用了一种无胶生物分析流程，包括耗尽的BAL蛋白的全蛋白质组消化，固定化金属离子亲和层析(IMAC)富集磷酸肽，线性离子陷阱质谱仪进行LC-MS/MS分析，以及搜索蛋白质序列数据库来生成一组BAL磷蛋白及其磷酸化位点。基于序列诊断MS/MS裂解模式，我们鉴定了36个磷酸化多肽的集合，包含26个不同的磷酸化位点。这些磷酸肽定位于21个磷酸蛋白，包括例如波形蛋白、纤溶蛋白-2、铁蛋白重链、激肽原-1等。表征的磷蛋白在细胞来源和生物学功能方面具有不同的特征。据我们所知，这项研究的结果代表了人类BAL磷酸蛋白质组的第一次描述。
Global-scale examination of protein phosphorylation in human biological fluids by phosphoproteomics approaches is an emerging area of research with potential for significant contributions towards discovery of novel biomarkers. In this pilot work, we analyzed the phosphoproteome in human bronchoalveolar lavage fluid (BAL) from nondiseased subjects. The main objectives were to assess the feasibility to probe phosphorylated proteins in human BAL and to obtain the initial catalog of BAL phosphoproteins, including protein identities and exact description of their phosphorylation sites. We used a gel-free bioanalytical workflow that included whole-proteome digestion of depleted BAL proteins, enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC), LC-MS/MS analyses with a linear ion trap mass spectrometer, and searches of a protein sequence database to generate a panel of BAL phosphoproteins and their sites of phosphorylation. Based on sequence-diagnostic MS/MS fragmentation patterns, we identified a collection of 36 phosphopeptides that contained 26 different phosphorylation sites. These phosphopeptides mapped to 21 phosphoproteins including, for example, vimentin, plastin-2, ferritin heavy chain, kininogen-1, and others. The characterized phosphoproteins have diverse characteristics in terms of cellular origin and biological function. To the best of our knowledge, results of this study represent the first description of the human BAL phosphoproteome.