Reproducible isolation of distinct, overlapping segments of the phosphoproteome
Reproducible isolation of distinct, overlapping segments of the phosphoproteome
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DOI：
10.1038/nmeth1005
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发表时间：
2007-03-01
期刊：
影响因子：
48
通讯作者：
Aebersold, Ruedi
中科院分区：
文献类型：
--
作者：
Bodenmiller, Bernd;Mueller, Lukas N.;Aebersold, Ruedi
常规分析和定量测量蛋白质组范围内蛋白质磷酸化变化的能力对于生物学和临床研究至关重要。我们评估了三种常见的磷酸肽分离方法（氨基磷酸酯化学（PAC），固定化金属亲和色谱（IMAC）和二氧化钛）的能力，可重复性，特异性和全面分离磷酸肽从复杂的混合物。从黑腹果蝇Kc 167细胞的胞质组分的胰蛋白酶消化物的等分试样中分离磷酸肽，并通过液相色谱-电喷雾电离串联质谱法进行分析。每种方法均可重复分离磷酸肽。然而，这些方法在分离的特异性方面不同，特别是在分离的磷酸肽组方面。结果表明，这三种方法检测不同的，部分重叠的片段的磷酸化蛋白质组，目前，没有一种方法是足够的一个全面的磷酸化蛋白质组分析。
The ability to routinely analyze and quantitatively measure changes in protein phosphorylation on a proteome-wide scale is essential for biological and clinical research. We assessed the ability of three common phosphopeptide isolation methods (phosphoramidate chemistry (PAC), immobilized metal affinity chromatography (IMAC) and titanium dioxide) to reproducibly, specifically and comprehensively isolate phosphopeptides from complex mixtures. Phosphopeptides were isolated from aliquots of a tryptic digest of the cytosolic fraction of Drosophila melanogaster Kc167 cells and analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Each method reproducibly isolated phosphopeptides. The methods, however, differed in their specificity of isolation and, notably, in the set of phosphopeptides isolated. The results suggest that the three methods detect different, partially overlapping segments of the phosphoproteome and that, at present, no single method is sufficient for a comprehensive phosphoproteome analysis.