CLIR-MS (cross-linking of isotope-labeled RNA and tandem mass spectrometry) allows for the identification for protein RNA interactions with single amino acid and nucleotide resolution. The Leitner, Aebersold, Hall and Allain groups present an extension of the CLIR-MS workflow during which the RNA is isotope labelled after crosslinking and thus, does not require segmentally labelled RNA. This workflow extension has been published in an article in Analytical Chemistry entitled "Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow".
Abstract
RNA–protein interactions mediate many intracellular processes. CLIR-MS (cross-linking of isotope-labeled RNA and tandem mass spectrometry) allows the identification of RNA–protein interaction sites at single nucleotide/amino acid resolution in a single experiment. Using isotopically labeled RNA segments for UV-light-induced cross-linking generates characteristic isotope patterns that constrain the sequence database searches, increasing spatial resolution. Whereas the use of segmentally isotopically labeled RNA is effective, it is technically involved and not applicable in some settings, e.g., in cell or tissue samples. Here we introduce an extension of the CLIR-MS workflow that uses unlabeled RNA during cross-linking and subsequently adds an isotopic label during sample preparation for MS analysis. After RNase and protease digests of a cross-linked complex, the nucleic acid part of a peptide–RNA conjugate is labeled using the enzyme T4 polynucleotide kinase and a 1:1 mixture of heavy 18O4-y-ATP and light ATP. In this simple, one-step reaction, three heavy oxygen atoms are transferred from the y-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.
Read the Publication in Analytical Chemistry (Open Acess)
Abstract, figure and title from Götze et al. (2021) Analytical Chemistry published under a CC BY-NC-ND 4.0 license.