Systematic Studies on TiO2-Based Phosphopeptide Enrichment Procedures upon in-Solution and in-Gel Digestions of Proteins. Are There Readily Applicable Protocols Suitable for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry-Based Phosphopeptide Stability Estimations?
Systematic Studies on TiO2-Based Phosphopeptide Enrichment Procedures upon in-Solution and in-Gel Digestions of Proteins. Are There Readily Applicable Protocols Suitable for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry-Based Phosphopeptide Stability Estimations?
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DOI：
10.1255/ejms.1134
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发表时间：
2011-10
影响因子：
1.3
通讯作者：
T. Eickner;S. Mikkat;P. Lorenz;M. Sklorz;R. Zimmermann;H. Thiesen;M. Glocker
中科院分区：
文献类型：
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作者：
T. Eickner;S. Mikkat;P. Lorenz;M. Sklorz;R. Zimmermann;H. Thiesen;M. Glocker
通过使用固定化金属亲和色谱（IMAC）和/或金属氧化物亲和色谱（MOAC），已经有许多成功的努力来富集复杂蛋白质混合物中的磷酸肽，其中磷酸肽的质谱分析已经成为专业实验室中的最新技术，主要应用基于nanoLC电喷雾电离质谱的研究。然而，这些强大的技术的广泛使用仍然没有实现。在这项研究中，我们提出了一个现成的磷酸肽富集程序，使用市售的二氧化钛加载的移液器吸头结合基质辅助激光解吸/电离质谱（MALDI-MS）分析。在样品加载期间使用β-酪蛋白作为模型蛋白质和柠檬酸作为添加剂，与应用2，5_二羟基苯甲酸（DHB）用于该任务相比，可以实现类似的富集成功。但是DHB继承的缺点被消除了。此外，我们表明，结合DHB和2，4，6-三羟基苯乙酮（THAP）作为基质的MALDI-MS测量保留了DHB的磷酸肽分析的灵敏度，但增加了THAP的均匀结晶性能，使MALDI-MS锚目标，自动MALDI-MS分析的先决条件，均匀分布的基质表面的准备。含三分基序的蛋白质28和stathmin是两个例子，其中成功的磷酸肽富集的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显示。最后，高分辨率MALDI傅立叶变换离子回旋共振质谱后磷酸肽富集表明，化学脱磷酸化可能发生的副反应，在基本洗脱磷酸肽绑定到MOAC表面，这表明蛋白质组范围的磷酸肽分析应谨慎解释。相比之下，深入分析磷酸化肽/非磷酸化肽兄弟姐妹可用于估计单个蛋白质中磷酸化位点的稳定性差异，可能增加生物调节过程的有价值的信息。
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 spectrometry-based investigations. However, widespread use of these powerful techniques is still not achieved. In this study, we present a ready-to-use phosphopeptide enrichment procedure using commercially available TiO2-loaded pipette tips in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. Using β-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 phosphopeptide/non-phosphorylated peptide siblings may be used to estimate stability differences of phosphorylation sites in individual proteins, possibly adding valuable information on biological regulation processes.