Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Rapid Commun Mass Spectrom. 2010 Mar;24(6):823-32. doi: 10.1002/rcm.4441.
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.
越来越多的人关注尿蛋白质组学，因为它有望发现各种疾病的生物标志物。然而，大多数尿蛋白质组学研究通常依赖于蛋白质预分级，到目前为止还没有对磷酸化状态进行特征描述。我们实验室最近开发了两种强大的方法，即集成多维液相色谱（IMDL）和阴阳多维液相色谱（MDLC）串联质谱法，具有高覆盖率的肽混合物鉴定。在这项研究中，我们采用了一种在蛋白质水平上不进行预分级的策略，分别使用 IMDL 和阴阳 MDLC 方法对肽进行分级，然后使用具有高分辨率和质量精度的线性离子阱-轨道阱（LTQ-Orbitrap）质谱仪进行鉴定。通过这两个实验，我们从两个实验中高度置信地鉴定了 1310 个非冗余蛋白质，其中显著包括 59 个磷酸化位点。超过一半注释的鉴定是膜相关蛋白。此外，还检测到溶酶体和肾脏相关蛋白。与之前发表的六个最大的尿蛋白质数据集相比，我们发现我们的数据包含了大部分已报道的蛋白质。我们的研究开发了一种探索人类尿蛋白质组的强大方法，这将提供一个全球性的尿蛋白质目录。据我们所知，这是首次对尿磷酸蛋白质组进行分析。这项工作显著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，该策略还可以作为生物标志物发现的参考。
