Immobilized gallium(III) affinity chromatography of phosphopeptides
Immobilized gallium(III) affinity chromatography of phosphopeptides
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DOI：
10.1021/ac981409y
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发表时间：
1999-07-15
影响因子：
7.4
通讯作者：
Tempst, P
中科院分区：
文献类型：
--
作者：
Posewitz, MC;Tempst, P
描述了一种用于微纯化磷酸化肽的新程序，作为质谱分析的前端。作为系统研究的结果，我们建议使用微尖固定金属亲和层析 (IMAC)（Erdjument-Bromage, H.；等人，J, Chromatogr., A 1998, 826, 167-181）形式，更具体地说与 Ga(III) 离子结合。手动 Ga(III) IMAC 易于执行；以近乎定量和高度选择性的方式回收磷酸肽，产生浓缩样品，用于通过基质辅助激光解吸/电离飞行时间和纳电喷雾电离质谱法进行直接分析。 Ga(Tn) 离子在选择性和多功能性（包括易于碱洗脱）方面比使用其他金属（例如 Fe(III) 和 Al(III)）具有明显的优势。选择性是最好的说明。通过有效富集磷酸化肽，这些磷酸化肽以类似于 2% 的摩尔比存在于非磷酸化蛋白质的背景上，这对于细胞中的蛋白质磷酸化状态来说可能是非常典型的情况。该系统还用于从人β4整联蛋白的胰蛋白酶消化物中检索并初步鉴定出五种以前未表征的磷酸肽，这些磷酸肽是通过免疫沉淀从细胞提取物中分离出来的。
A novel procedure for micropurification of phosphorylated peptides, as a front end to mass spectrometric analysis, is described. As a result of a systematic study, we propose the use of an immobilized metal affinity chromatography (IMAC) in a microtip (Erdjument-Bromage, H.; et al, J, Chromatogr., A 1998, 826, 167-181) format, more specifically in combination with Ga(III) ions. Manual Ga(III) IMAC is easy to perform; phosphopeptides are retrieved in a near-quantitative and highly selective manner, to yield a concentrated sample for direct analysis by matrix-assisted laser desorption/ionization time-of-flight and nanoelectrospray ionization mass spectrometry. Ga(Tn) ions offer distinct advantages over the use of other metals, such as Fe(III) and Al(III) in terms of both selectivity and versatility, including facile base elution. Selectivity is best illustrated. by effective enrichment of phosphopeptides that were present in a molar ratio of similar to 2% on a background of umphosphorylated protein, a situation very typical perhaps for protein phosphorylation states in the cell. The system was also used to retrieve and tentatively identify five previously uncharacterized phosphopeptides from a tryptic digest of human beta 4 integrin, isolated from cell extracts by immunoprecipitation.