Josef Maryáš1,2, Jakub Faktor1,2, Lenka Čápková1, Petr Müller2, Petr Skládal1, Pavel Bouchal3. 1. Masaryk University, Faculty of Science, Department of Biochemistry, Brno, Czech Republic. 2. Masaryk Memorial Cancer Institute, Regional Centre for Applied Molecular Oncology, Brno, Czech Republic. 3. Masaryk University, Faculty of Science, Department of Biochemistry, Brno, Czech Republic bouchal@chemi.muni.cz.
Abstract
BACKGROUND/AIM: Pul-down assay is a popular in vitro method for identification of physical interactors of selected proteins. Here, for the first time, we compared three conventional variants of pull-down assay with the streptavidin-modified surface plasmon resonance (SPR) chips for the detection of PDZ and LIM domain protein 2 (PDLIM2) interaction partners. MATERIALS AND METHODS: PDLIM2 protein-protein interactions were analysed by three variants of pull-down assay on streptavidin beads using LC-MS/MS in "Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH)" mode and compared with LC-SWATH-MS/MS data from SPR chips. RESULTS: The results showed that (i) the use of SPR chip led to comparable data compared to on-column streptavidin beads, (ii) gravity flow and microflow in wash and elution steps provided better results than centrifugation, and (iii) type and concentration of detergent did not significantly affect the interactome data of cancer-associated PDLIM2. CONCLUSION: Our study supports further application of SPR-based affinity purification with SWATH mass spectrometry for reproducible and controlled characterization of cancer-associated interactomes. Copyright
BACKGROUND/AIM: Pul-down assay is a popular in vitro method for identification of physical interactors of selected proteins. Here, for the first time, we compared three conventional variants of pull-down assay with the streptavidin-modified surface plasmon resonance (SPR) chips for the detection of PDZ and LIM domain protein 2 (PDLIM2) interaction partners. MATERIALS AND METHODS:PDLIM2 protein-protein interactions were analysed by three variants of pull-down assay on streptavidin beads using LC-MS/MS in "Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH)" mode and compared with LC-SWATH-MS/MS data from SPR chips. RESULTS: The results showed that (i) the use of SPR chip led to comparable data compared to on-column streptavidin beads, (ii) gravity flow and microflow in wash and elution steps provided better results than centrifugation, and (iii) type and concentration of detergent did not significantly affect the interactome data of cancer-associated PDLIM2. CONCLUSION: Our study supports further application of SPR-based affinity purification with SWATH mass spectrometry for reproducible and controlled characterization of cancer-associated interactomes. Copyright
Authors: Pavel Bouchal; Monika Dvořáková; Theodoros Roumeliotis; Zbyněk Bortlíček; Ivana Ihnatová; Iva Procházková; Jenny T C Ho; Josef Maryáš; Hana Imrichová; Eva Budinská; Rostislav Vyzula; Spiros D Garbis; Bořivoj Vojtěšek; Rudolf Nenutil Journal: Mol Cell Proteomics Date: 2015-04-22 Impact factor: 5.911