A qualitative and quantitative framework for the characterization of protein-RNA interactions by cross-linking mass spectrometry (XL-MS)
- 1ETH Zurich
Abstract
Protein-RNA interactions regulate nearly all levels of gene expression. Their characterization at the molecular basis is critical for understanding the function of protein-RNA complexes, both in cell homeostasis and pathology. Yet, molecular interactions underlying the formation of these complexes remain understudied. UV cross-linking mass spectrometry (XL-MS) is one of the leading approaches for in vitro identification of protein-RNA contact sites, and for providing structural information in the form of distance constraints crucial for the refinement of structural models. Our previously published CLIR-MS method enables qualitative characterization of protein-RNA interactions at residue-specific resolution but was not designed for highly accurate quantitative interpretation. Here, we present a XL-MS workflow based on irradiation with UV light that combines data-dependent acquisition (DDA) and parallel reaction monitoring (PRM) for simultaneous detection and characterization of protein-RNA interaction sites at residue-specific resolution, followed by label-free quantification of cross-linked products. We successfully apply our combined workflow to in vitro reconstituted protein-RNA complexes formed by the proteins TDP-43 and FOX1. We report on novel RNA interaction sites located in the oligomerization interface of the N-terminal domain (NTD) of TDP-43, and characterize quantitatively the process of RNA-induced monomerization of the NTD in an RNA titration experiment. We also investigate temperature-dependent differences in the efficiency of cross-link formation in the FOX1 complex. Our framework offers a unique and novel approach to comparative quantification of protein-RNA cross-links, overcoming the limitations imposed by conventional UV XL-MS methods.
Related
- CRISPR-dPCR: A unified multiplex digital PCR framework for quantitative characterization of targeted knock-in
- ProtBed: a streamlined web platform for exploratory statistics and predictive modeling in quantitative mass spectrometry
- Off-line LCxLC-Mass Spectrometry Workflow using CID and EAD for the Quantification and Comprehensive Structural Double Bond Characterization of Glycerophospholipids in Plasma Samples
- Hydrophilic interaction chromatography and tandem mass spectrometry for the characterization of methylated oligoribonucleotides
- Quantitative Characterization of RNA-Splinted Ligation as a Multipurpose Post-Synthetic Strategy for High-Density DNA Microarrays Synthesized by Nucleic Acid Photolithography.
BibTeX
@misc{smok2026qualitative,
title = {{A qualitative and quantitative framework for the characterization of protein-RNA interactions by cross-linking mass spectrometry (XL-MS)}},
author = {I. Smok and A. Zbinden and J. Osterroth and J. Jiang and F. H.- T. Allain and J. Hall and M. Polymenidou and A. Leitner},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.15.756239},
url = {https://www.biorxiv.org/content/10.64898/2026.10.15.756239v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - A qualitative and quantitative framework for the characterization of protein-RNA interactions by cross-linking mass spectrometry (XL-MS)
AU - I. Smok
AU - A. Zbinden
AU - J. Osterroth
AU - J. Jiang
AU - F. H.- T. Allain
AU - J. Hall
AU - M. Polymenidou
AU - A. Leitner
PY - 2026
DA - 2026/10/15
PB - bioRxiv
DO - 10.64898/2026.10.15.756239
UR - https://www.biorxiv.org/content/10.64898/2026.10.15.756239v1
AB - Protein-RNA interactions regulate nearly all levels of gene expression. Their characterization at the molecular basis is critical for understanding the function of protein-RNA complexes, both in cell homeostasis and pathology. Yet, molecular interactions underlying the formation of these complexes remain understudied. UV cross-linking mass spectrometry (XL-MS) is one of the leading approaches for in vitro identification of protein-RNA contact sites, and for providing structural information in the form of distance constraints crucial for the refinement of structural models. Our previously published CLIR-MS method enables qualitative characterization of protein-RNA interactions at residue-specific resolution but was not designed for highly accurate quantitative interpretation. Here, we present a XL-MS workflow based on irradiation with UV light that combines data-dependent acquisition (DDA) and parallel reaction monitoring (PRM) for simultaneous detection and characterization of protein-RNA interaction sites at residue-specific resolution, followed by label-free quantification of cross-linked products. We successfully apply our combined workflow to in vitro reconstituted protein-RNA complexes formed by the proteins TDP-43 and FOX1. We report on novel RNA interaction sites located in the oligomerization interface of the N-terminal domain (NTD) of TDP-43, and characterize quantitatively the process of RNA-induced monomerization of the NTD in an RNA titration experiment. We also investigate temperature-dependent differences in the efficiency of cross-link formation in the FOX1 complex. Our framework offers a unique and novel approach to comparative quantification of protein-RNA cross-links, overcoming the limitations imposed by conventional UV XL-MS methods.
ER -