title: Pulse Proteolysis and Precipitation for Target Identification.
abstract: 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.
authors.affiliation: Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) , 6681/92-A. Av. Ipiranga-TECNOPUC-Prédio 92A 90619-900, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Rogério V
authors.initials: RV
authors.lastname: Trindade
authors.affiliation: Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) , 6681/92-A. Av. Ipiranga-TECNOPUC-Prédio 92A 90619-900, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Antônio F M
authors.initials: AF
authors.lastname: Pinto
authors.affiliation: Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) , 6681/92-A. Av. Ipiranga-TECNOPUC-Prédio 92A 90619-900, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Diógenes S
authors.initials: DS
authors.lastname: Santos
authors.affiliation: Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS) , 6681/92-A. Av. Ipiranga-TECNOPUC-Prédio 92A 90619-900, Porto Alegre, Rio Grande do Sul, Brazil.
authors.forename: Cristiano V
authors.initials: CV
authors.lastname: Bizarro
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis; D011232:Chemical Precipitation; D055808:Drug Discovery / Q000379:methods*; D008024:Ligands; D020102:Mycobacterium smegmatis / Q000737:chemistry; D009169:Mycobacterium tuberculosis / Q000737:chemistry; D059748:Proteolysis
chemical_list: C090796:ATP-binding protein, bacteria; D001426:Bacterial Proteins; D002352:Carrier Proteins; D008024:Ligands
keywords: PePTID; energetics-based target discovery approach; pulse proteolysis; target discovery