Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis
Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis
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DOI：
10.1002/pmic.200600045
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发表时间：
2007-02-01
期刊：
影响因子：
3.4
通讯作者：
Zou, Hanfa
中科院分区：
文献类型：
--
作者：
Feng, Shun;Pan, Chensong;Zou, Hanfa
固定化金属亲和层析(IMAC)是一种常用的磷酸蛋白质组分析技术，因为它对磷酸肽的吸附具有很高的亲和力。IMAC柱的小型化对于少量样品的分析是必不可少的。用亚氨基二乙酸(IDA)化学修饰硅胶整体柱，然后将Fe3+离子固定在毛细管内，制备了纳米IMAc柱VAS。用MALDI-TOF MS分析表明，Fe3+-IDA硅胶IMAC整体毛细管柱可以从酪蛋白的胰酶解液中特异地捕获磷酸肽。硅胶整体IMAC毛细管柱与纳米流RPLC/纳米螺旋ESI质谱仪(MU RPLC-NanESI MS)手动联用进行磷酸蛋白质组分析。通过对标准磷蛋白的分析验证了该体系的有效性，并将其应用于小鼠肝脏裂解液中蛋白质的磷酸化分析。除了MS/MS谱图外，还收集了中性损失峰的MS/MS/MS谱图。经过数据库搜索和保守标准的人工验证，通过分析仅12微克的小鼠肝脏裂解物的胰酶消化，鉴定出29个单磷酸化肽。结果表明，硅胶整体IMAC毛细管色谱柱与gRPLC-NanESI MS联用非常适合于微量样品的磷蛋白质组分析。
Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column vas prepared by chemical m6dification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. it was demonstrated that Fe3+-IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of (x-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (mu RPLC-nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12 mu g mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with gRPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample.