Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry
Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry
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DOI：
10.1074/mcp.t300006-mcp200
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发表时间：
2003-11-01
影响因子：
7
通讯作者：
Peck, SC
中科院分区：
文献类型：
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作者：
Nühse, TS;Stensballe, A;Peck, SC
由于磷蛋白通常含量较低，对蛋白质磷酸化进行全局分析需要特定的富集方法。到目前为止，用于磷酸肽的固定化金属离子亲和色谱（IMAC）在大规模研究中显示出很大的潜力，但特异性较差是其众所周知的问题。我们研究了IMAC与毛细管液相色谱 - 串联质谱联用用于鉴定拟南芥质膜磷蛋白的潜力。在不进行肽段化学修饰的情况下，从质膜消化物中分离出纯度超过75%的磷酸肽，并通过质谱进行检测和测序。我们提出了一种在IMAC之前使用强阴离子交换色谱进行二维肽段分离的方案，该方案既降低了IMAC纯化的磷酸肽的复杂性，又能更广泛地覆盖单磷酸化肽段。在已鉴定的序列中，有六个来自质膜H⁺ - ATP酶的不同异构体，并在调节性C末端确定了两个先前未知的磷酸化位点。大规模鉴定质膜蛋白磷酸化位点的潜力将对信号转导、细胞间通讯和膜转运过程的研究产生广泛影响。
Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poor specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides. Among the identified sequences, six originated from different isoforms of the plasma membrane H+-ATPase and defined two previously unknown phosphorylation sites at the regulatory C terminus. The potential for large-scale identification of phosphorylation sites on plasma membrane proteins will have wide-ranging implications for research in signal transduction, cell-cell communication, and membrane transport processes.