Displacers improve the selectivity of phosphopeptide enrichment by metal oxide affinity chromatography

Boletín Médico Del Hospital Infantil de México (English Edition) Pub Date : 2017-05-01 DOI:10.1016/j.bmhime.2017.11.028

Yesenia Herrera , Sandra Contreras , Magdalena Hernández , Laura Álvarez , Yolanda Mora , Sergio Encarnación-Guevara

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Abstract

Background

A key process in cell regulation is protein phosphorylation, which is catalyzed by protein kinases and phosphatases. However, phosphoproteomics studies are difficult because of the complexity of protein phosphorylation and the number of phosphorylation sites.

Methods

We describe an efficient approach analyzing phosphopeptides in single, separated protein by two-dimensional gel electrophoresis. In this method, a titanium oxide (TiO₂)-packed NuTip is used as a phosphopeptide trap, together with displacers as lactic acid in the loading buffer to increase the efficiency of the interaction between TiO₂ and phosphorylated peptides.

Results

The results were obtained from the comparison of mass spectra of proteolytic peptides of proteins with a matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) instrument.

Conclusions

This method has been applied to identifying phosphoproteins involved in the symbiosis Rhizobium etli-Phaseolus vulgaris.

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取代剂提高了金属氧化物亲和层析富集磷酸肽的选择性

细胞调控的一个关键过程是蛋白磷酸化，这是由蛋白激酶和磷酸酶催化的。然而，由于蛋白质磷酸化的复杂性和磷酸化位点的数量，磷酸化蛋白质组学研究是困难的。方法采用双向凝胶电泳技术，对单个分离蛋白中的磷酸肽进行分析。在该方法中，使用氧化钛(TiO2)填充的NuTip作为磷酸肽陷阱，并在负载缓冲液中使用置换剂作为乳酸，以提高TiO2与磷酸化肽之间相互作用的效率。结果用基质辅助激光解吸-电离飞行时间(MALDI-TOF)仪对蛋白质的蛋白水解肽质谱进行了比较。结论该方法可用于鉴定菜豆根瘤菌与菜豆根瘤菌共生的磷酸化蛋白。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Boletín Médico Del Hospital Infantil de México (English Edition)

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