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首页> 外文期刊>Biochemistry >Kinase Substrate Profiling Using a Proteome-wide Serine-Oriented Human Peptide Library
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Kinase Substrate Profiling Using a Proteome-wide Serine-Oriented Human Peptide Library

机译：激酶底物分析使用蛋白质型丝氨酸取向的人肽文库

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摘要

The human proteome encodes 500 protein kinases and hundreds of thousands of potential phosphorylation sites. However, the identification of kinase-substrate pairs remains an active area of research because the relationships between individual kinases and these phosphorylation sites remain largely unknown. Many techniques have been established to discover kinase substrates but are often technically challenging to perform. Moreover, these methods frequently rely on substrate reagent pools that do not reflect human protein sequences or are biased by human cell line protein expression profiles. Here, we describe a new approach called SERIOHL-KILR (serine-oriented human library-kinase library reactions) to profile kinase substrate specificity and to identify candidate substrates for serine kinases. Using a purified library of 100000 serine-oriented human peptides expressed heterologously in Escherichia coli, we perform in vitro kinase reactions to identify phosphorylated human peptide sequences by liquid chromatography and tandem mass spectrometry. We compare our results for protein kinase A to those of a well-established positional scanning peptide library method, certifying that SERIOHL-KILR can identify the same predominant motif elements as traditional techniques. We then interrogate a small panel of cancer-associated PKC beta mutants using our profiling protocol and observe a shift in substrate specificity likely attributable to the loss of key polar contacts between the kinase and its substrates. Overall, we demonstrate that SERIOHL-KILR can rapidly identify candidate kinase substrates that can be directly mapped to human sequences for pathway analysis. Because this technique can be adapted for various kinase studies, we believe that SERIOHL-KILR will have many new victims in the future.
机译：人蛋白质组编码＆ 500蛋白激酶和数百种潜在的磷酸化位点。然而，激酶基质对的鉴定仍然是活跃的研究领域，因为单个激酶与这些磷酸化位点之间的关系仍然很大程度上是未知的。已经建立了许多技术来发现激酶基材，但经常在技术上具有挑战性。此外，这些方法经常依赖于不反映人蛋白序列或被人细胞系蛋白表达谱偏置的底物试剂池。在此，我们描述了一种称为SerioHl-Kilr（丝氨酸人库 - 激酶库反应）的新方法，以概况激酶底物特异性，并鉴定丝氨酸激酶的候选底物。使用纯化的＆ 100000丝氨酸的人肽在大肠杆菌中表达异构，我们通过液相色谱和串联质谱法进行体外激酶反应以鉴定磷酸化的人肽序列。我们将结果与蛋白激酶A的结果进行比较，以确定良好的位置扫描肽库方法，证明SerioHL-Kilr可以识别与传统技术相同的主要基序元素。然后，我们使用我们的分析方案询问小型癌症相关的PKCβ突变体，并观察底物特异性的偏移，可能归因于激酶和其基材之间的关键极性触点的损失。总的来说，我们证明SerioHl-Kilr可以快速识别可以直接映射到人体序列的候选激酶底物。因为这种技术可以适用于各种激酶研究，所以我们认为Seriohl-Kilr将来会有许多新的受害者。

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  • 来源
    《Biochemistry》 |2018年第31期|共9页
  • 作者

    Barber Karl W.; Miller Chad J.; Jun Jay W.; Lou Hua Jane; Turk Benjamin E.; Rinehart Jesse;

  • 作者单位

    Yale Univ Dept Cellular &

    Mol Physiol New Haven CT 06520 USA;

    Yale Univ Dept Pharmacol New Haven CT 06520 USA;

    Cornell Univ Div Nutr Sci Ithaca NY 14850 USA;

    Yale Univ Dept Pharmacol New Haven CT 06520 USA;

    Yale Univ Dept Pharmacol New Haven CT 06520 USA;

    Yale Univ Dept Cellular &

    Mol Physiol New Haven CT 06520 USA;

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  • 正文语种 eng
  • 中图分类 生物化学;
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