Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry
Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry
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DOI：
10.1002/rcm.4377
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发表时间：
2010-01-01
影响因子：
2
通讯作者：
Ross, Andrew R. S.
中科院分区：
文献类型：
--
作者：
Aryal, Uma K.;Ross, Andrew R. S.
二氧化钛金属氧化物亲和色谱（TiO 2-MOAC）被广泛认为是比固定化金属离子亲和色谱（IMAC）更有选择性的磷酸肽富集。然而，TiO 2-MOAC的生物样品的广泛应用受到了相互矛盾的报告，其中的实验条件是最佳的。我们已经评估了TiO 2-MOAC在宽范围的负载和洗脱条件下的性能。用强酸性溶液装载和严格洗涤肽确保了对磷酸化肽的高度选择性富集，并且非磷酸化肽的残留最小。与以前的报道相反，发现在上样溶液中加入乙醇酸会降低对磷酸肽的特异性。氢氧化铵或磷酸铵中的碱洗脱提供了磷酸化肽的最佳特异性和回收率。相反，用磷酸洗脱得到磷酸肽的不完全回收，而在洗脱液中包含2，5-二羟基苯甲酸引入了对多重磷酸化肽的回收的偏倚。还发现TiO 2-MOAC对蛋白质纯化过程中常用作磷酸酶抑制剂的许多试剂不耐受。然而，TiO 2-MOAC表现出更高的特异性比固定化镓（Ga 3+），固定化铁（Fe 3+），或二氧化锆（ZrO 2）亲和层析磷酸肽富集。基质辅助激光解吸/电离质谱（MALDI-MS）更有效地检测较大的，多磷酸化的肽比液相色谱/电喷雾串联质谱（LC/ESI-MS/MS），这是更有效地为较小的，单磷酸化的肽。版权所有（C）2009皇冠在加拿大的权利。由John Wiley & Sons有限公司出版
Titanium dioxide metal oxide affinity chromatography (TiO2-MOAC) is widely regarded as being more selective than immobilized metal-ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO2-MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO2-MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non-phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5-dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO2-MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO2-MOAC showed higher specificity than immobilized gallium (Ga3+), immobilized iron (Fe3+), or zirconium dioxide (ZrO2) affinity chromatography for phosphopeptide enrichment. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS), which was more efficient for smaller, singly phosphorylated peptides. Copyright (C) 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.