Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow
Michael Götze, Chris P. Sarnowski, Tebbe de Vries, Anna Knörlein, Frédéric H.‐T. Allain, Jonathan Hall, Ruedi Aebersold, Alexander Leitner
Abstract
-γ-ATP and light ATP. In this simple, one-step reaction, three heavy oxygen atoms are transferred from the γ-phosphate to the 5'-end of the RNA, introducing an isotopic shift of 6.01 Da that is detectable by mass spectrometry. We applied this approach to the RNA recognition motif (RRM) of the protein FOX1 in complex with its cognate binding substrate, FOX-binding element (FBE) RNA. We also labeled a single phosphate within an RNA and unambiguously determined the cross-linking site of the FOX1-RRM binding to FBE at single residue resolution on the RNA and protein level and used differential ATP labeling for relative quantification based on isotope dilution. Data are available via ProteomeXchange with the identifier PXD024010.