Pulse Proteolysis and Precipitation for Target Identification

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• 作者：Rogério V. Trindade ; Antônio F. M. Pinto ; Diógenes S. Santos ; Cristiano V. Bizarro
• 刊名：Journal of Proteome Research
• 出版年：2016
• 出版时间：July 1, 2016
• 年：2016
• 卷：15
• 期：7
• 页码：2236-2245
• 全文大小：576K
• 年卷期：0
• ISSN：1535-3907

文摘

In recent years, phenotypic screening has assumed a leading role in drug discovery efforts. However, development of new drugs from bioactive compounds obtained in screening campaigns requires identification of the cellular targets responsible for their biological activities. A new energetics-based method for target identification is presented: pulse proteolysis and precipitation for target identification (PePTID). In this method, proteins incubated with or without a ligand and submitted to a brief proteolytic pulse are directly analyzed and compared using a label-free semiquantitative mass spectrometry strategy, dispensing the SDS-PAGE readout and greatly improving the throughput. As a proof-of-concept, we applied the PePTID method to identify ATP-binding proteins in Mycobacterium smegmatis, a model system for Mycobacterium tuberculosis, the etiological agent of tuberculosis.