Yongmei Li*, Baicai Yang, Yali Zhang, Kaiwen Hei and Mingming Xiao Pages 1 - 7 ( 7 )
Background To investigate the interactions between RNA and proteins is essential to understand how these macromolecule complexes exert their functions. RNA pull-down is a classic technique to enrich RNA binding proteins, however, a large number of non-specific binding proteins may be enriched during sample preparation, interfering with the downstream mass spectrometric analyses and also causing false positives.
Objective In this study we examined the background contaminates in RNA pull-down experiment using mass spectrometric analysis. Method Antisense MALAT1 was first synthesized using in vitro transcription and incubated with cellular proteins extracted from HepG2 cells. The non-specific binding proteins were isolated using streptavidin conjugated magnetic beads and separated on SDS-PAGE. Each gel lane was divided into nine bands and digested with trypsin for the downstream LC-MS/MS analyses.
Results: 191 protein groups were identified as non-specific binding proteins in RNA pull-down samples. In addition, comparison between different sample preparation conditions showed that the level of background contaminates were mostly induced by the solid phase support and not affected by the studied RNA. In addition, using more stringent detergent and streptavidin magnetic beads with smaller size could reduce the amount of background interfering proteins.
Conclusion: This study provides a reference to distinguish bona fide RNA interacting proteins from the background contaminants. The results also demonstrate that different sample preparation conditions have great impacts on the level of enriched background contaminates, shedding new light on the optimization of RNA pull-down experiment.
LC-MS/MS; RNA pull-down; RNA-protein interaction; SDS-PAFE, contaminates
Tianjin Medical University, Department of Pathogen Biology, Tianjin Medical University, Department of Pathogen Biology, Tianjin Medical University, Department of Pathogen Biology, Tianjin Medical University, Department of Pathogen Biology, Tianjin Medical University, Department of Pathogen Biology