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.
Methods: 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, a comparison between different sample preparation conditions showed that
the level of background contaminates was mostly induced by the solid phase support rather than
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 experiments.