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首页> 外文期刊>European journal of mass spectrometry >Systematic studies on TiO_2-based phosphopeptide enrichment procedures upon in-solution and in-gel digestions of roteins. Are there readily applicable protocols suitable for matrix-assisted laser desorption/ionization mass spectrometrybased phosphopeptide stability estimations?
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Systematic studies on TiO_2-based phosphopeptide enrichment procedures upon in-solution and in-gel digestions of roteins. Are there readily applicable protocols suitable for matrix-assisted laser desorption/ionization mass spectrometrybased phosphopeptide stability estimations?

机译：对蛋白质进行溶液内和凝胶内消化后，对基于TiO_2的磷酸肽富集程序的系统研究。是否存在适用于基于基质辅助激光解吸/电离质谱的磷酸肽稳定性评估的适用方案？

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There have been many successful efforts to enrich phosphopeptides in complex protein mixtures by the use of immobilized metal affinity chromatography (IMAC) and/or metal oxide affinity chromatography (MOAC) with which mass spectrometric analysis of phosphopeptides has become state of the art in specialized laboratories, mostly applying nanoLC electrospray ionization mass spectrometrybased investigations. However, widespread use of these powerful techniques is still not achieved. In this study, we present a ready-touse phosphopeptide enrichment procedure using commercially available TiO _2-loaded pipette tips in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. Using b-casein as a model protein and citric acid as additive during sample loading, a similar enrichment success can be achieved as compared to applying 2,5-dihydroxy benzoic acid (DHB) for this task. But the DHB-inherited drawbacks are eliminated. In addition, we show that combining DHB and 2,4,6-trihydroxy acetophenone (THAP) as matrix for MALDI-MS measurements retains the sensitivity of DHB for phosphopeptide analysis but adds the homogenous crystallization properties of THAP, enabling preparation of evenly distributed matrix surfaces on MALDI-MS anchor targets, a prerequisite for automated MALDI-MS analyses. Tripartite motif-containing protein 28 and stathmin are two examples for which successful phosphopeptide enrichment of either sodium dodecyl sulfate polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis- separated proteins is shown. Finally, high resolution MALDI Fourier transform ion cyclotron resonance mass spectrometry after phosphopeptide enrichment suggests that chemical dephosphorylation may occur as a side reaction during basic elution of phosphopeptides bound to MOAC surfaces, suggesting that proteome-wide phosphopeptide analyses ought to be interpreted with caution. In contrast, in-depth analysis of phosphopeptideon-phosphorylated peptide siblings may be used to estimate stability differences of phosphorylation sites in individual proteins, possibly adding valuable information on biological regulation processes.
机译：通过固定化金属亲和色谱（IMAC）和/或金属氧化物亲和色谱（MOAC），已经进行了许多成功的努力来富集复杂蛋白混合物中的磷酸肽，在专用实验室中，磷酸肽的质谱分析已成为最新技术，主要应用基于nanoLC电喷雾电离质谱的研究。但是，仍然没有广泛使用这些强大的技术。在这项研究中，我们提出了一种现成的磷酸肽富集程序，该程序使用了可购得的装有TiO _2的移液器吸头，并结合了基质辅助的激光解吸/电离质谱（MALDI-MS）分析。与在样品上样过程中使用b-酪蛋白作为模型蛋白质和柠檬酸作为添加剂相比，将2，5-二羟基苯甲酸（DHB）应用于此任务可实现类似的富集成功。但是消除了DHB继承的缺点。此外，我们显示将DHB和2,4,6-三羟基苯乙酮（THAP）组合作为基质进行MALDI-MS测量可保留DHB对磷酸肽分析的敏感性，但增加了THAP的均质结晶特性，从而可以制备均匀分布的基质MALDI-MS锚定目标上的曲面，这是自动MALDI-MS分析的前提。包含三方基序的蛋白质28和stathmin是两个实例，显示了十二烷基硫酸钠聚丙烯酰胺凝胶电泳或二维凝胶电泳分离的蛋白质的成功磷酸肽富集。最后，磷酸肽富集后的高分辨率MALDI傅里叶变换离子回旋共振质谱表明，在与MOAC表面结合的磷酸肽的基本洗脱过程中，化学去磷酸化可能会作为副反应发生，这表明蛋白质组范围的磷酸肽分析应谨慎解释。相反，对磷酸肽/非磷酸化肽同胞的深入分析可用于估计单个蛋白质中磷酸化位点的稳定性差异，可能会增加有关生物学调控过程的有价值的信息。

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