Toward universal protein post-translational modification detection in high throughput format,Chemical Communications

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Toward universal protein post-translational modification detection in high throughput format
Chemical Communications ( IF 4.3 ) Pub Date : 2018-03-02 00:00:00 , DOI: 10.1039/c7cc09575a
Harri Härmä _{1,

2,

3} , Natalia Tong-Ochoa _{1,

2,

3} , Arjan J. van Adrichem _{3,

4,

5} , Ilian Jelesarov _{6,

7,

8} , Krister Wennerberg _{3,

4,

5} , Kari Kopra _{1,

2,

3}

Affiliation

Post-translational modification (PTM) of proteins plays essential regulatory roles in a variety of pathological conditions. Reliable and practical assays are required to accelerate the discovery of inhibitors and activators for PTM related diseases. Today, methodologies are based on specific or group-specific PTM recognition of e.g. phosphate for kinase activity without extending to other type of PTMs. Here we have established a universal time-resolved luminescence assay on a peptide-break platform for the direct detection of wide variety of PTMs. The developed assay is based on the leucine zipper concept wherein a europium-chelate labeled detection peptide and a non-labeled peptide substrate form a highly luminescent dimer. As an active PTM enzyme at sub or low nanomolar concentration modifies the substrate peptide, the luminescent signal of the detached detection peptide is quenched in the presence of soluble quenchers. The functionality of this universal assay technique has been demonstrated for the monitoring of phosphorylation, dephosphorylation, deacetylation, and citrullination with high applicability also to other PTMs in a high throughput format.

中文翻译：

迈向高通量格式的通用蛋白质翻译后修饰检测

蛋白质的翻译后修饰（PTM）在多种病理条件下均起着重要的调节作用。需要可靠且实用的分析方法来加快发现PTM相关疾病的抑制剂和活化剂的速度。如今，方法论是基于特定或特定组的PTM识别，例如磷酸可增强激酶活性，而不会扩展到其他类型的PTM。在这里，我们在肽断裂平台上建立了通用的时间分辨发光测定法，用于直接检测多种PTM。开发的测定法基于亮氨酸拉链概念，其中a螯合物标记的检测肽和未标记的肽底物形成高发光二聚体。当亚摩尔浓度或低纳摩尔浓度的活性PTM酶修饰底物肽时，分离的检测肽的发光信号在可溶性淬灭剂的存在下被淬灭。已经证明了这种通用测定技术的功能性，可用于监测磷酸化，去磷酸化，脱乙酰化和瓜氨酸化，并且对高通量格式的其他PTM也具有很高的适用性。

更新日期：2018-03-15

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