A comprehensive and non-prefractionation on the protein level approach for the human urinary proteome: touching phosphorylation in urine
A comprehensive and non-prefractionation on the protein level approach for the human urinary proteome: touching phosphorylation in urine
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DOI：
10.1002/rcm.4441
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发表时间：
2010-03-01
影响因子：
2
通讯作者：
Zeng, Rong
中科院分区：
文献类型：
--
作者：
Li, Qing-Run;Fan, Ke-Xin;Zeng, Rong
由于尿蛋白质组有望发现各种疾病的生物标志物，因此越来越受到人们的关注。然而，大多数尿蛋白质组学研究通常依赖于蛋白质预分离，迄今为止没有呈现磷酸化状态的表征。两个强大的方法，集成多维液相色谱（IMDL）和阴阳多维液相色谱（MDLC）串联质谱，最近在我们的实验室，开发了高覆盖识别肽混合物。在这项研究中，我们采用了一种策略，没有预先分馏的蛋白质水平上的尿蛋白质组鉴定，使用IMDL和阴阳MDLC方法的肽分级，然后使用线性离子阱轨道阱（LTQ-Orbitrap）质谱仪鉴定具有高分辨率和质量准确度。从两个实验中高度自信地鉴定了总共1310个非冗余蛋白质，显著地包括59个磷酸化位点。超过一半的注释鉴定是膜相关蛋白。此外，还检测到溶酶体以及肾相关蛋白。与以往发表的六个最大的尿蛋白数据集相比，我们发现我们的数据包含了大多数已报道的蛋白质。我们的研究开发了一个强大的方法来探索人类尿蛋白质组，这将提供一个全球范围内的尿蛋白目录。据我们所知，这是第一个报告，以描绘尿磷酸蛋白质组。这项工作大大扩展了目前对尿蛋白修饰及其潜在生物学意义的理解。此外，该策略可以进一步作为生物标志物发现的参考。版权所有（C）2010约翰威利冷杉父子有限公司
Increasing attention has been paid to the urinary proteome because it holds the promise of discovering various disease biomarkers. However, most of the urine proteomics studies routinely relied on protein pre-fractionation and so far did not present characterization on phosphorylation status. Two robust approaches, integrated multidimensional liquid chromatography (IMDL) and Yin-yang multidimensional liquid chromatography (MDLC) tandem mass spectrometry, were recently developed in our laboratory, with high-coverage identification of peptide mixtures. In this study, we adopted a strategy without pre-fractionation on the protein level for urinary proteome identification, using both the IMDL and the Yin-yang MDLC methods for peptide fractionation followed by identification using a linear ion trap-orbitrap (LTQ-Orbitrap) mass spectrometer with high resolution and mass accuracy. A total of 1310 non-redundant proteins were highly confidently identified from two experiments, significantly including 59 phosphorylation sites. More than half the annotated identifications were membrane-related proteins. In addition, the lysosomal as well as kidney-associated proteins were detected. Compared with the six largest datasets of urinary proteins published previously, we found our data included most of the reported proteins. Our study developed a robust approach for exploring the human urinary proteome, which would provide a catalogue of urine proteins on a global scale. It is the first report, to our best knowledge, to profile the urinary phosphoproteome. This work significantly extends current comprehension of urinary protein modification and its potential biological significance. Moreover, the strategy could further serve as a reference for biomarker discovery. Copyright (C) 2010 John Wiley fir Sons, Ltd.