Trapped-ion mobility spectrometry with PASEF for analysis of RNA-protein crosslinks in Escherichia coli ribosomes and HeLa cell line nucleic extract

Covalent crosslinking through UV exposure or chemical agents is a method used to analyze the interaction between nucleic acids and proteins. This method involves RNA-protein crosslinking, followed by the analysis of the resulting nucleotide-peptide crosslinks using high-resolution mass spectrometry. It can be applied on a proteome-wide scale to discover new RNA-binding proteins, or on isolated macromolecular complexes to complement unresolved structures determined by other structural methods. However, reliably identifying and quantifying nucleotide-peptide crosslinks using mass spectrometry remains challenging. We demonstrate the use of trapped-ion mobility spectrometry in the timsTOF mass spectrometer, first in the dda-PASEF mode to characterize ion mobility of the crosslinks, and then in the dia-PASEF mode with a customized spectral library to quantify the crosslinks. We conducted crosslinking experiments using E. coli ribosomes and HeLa nucleic extract, with both samples crosslinked using UV 254 nm and ribosomes additionally using nitrogen mustard chemical (NM). The samples were treated with nucleases and trypsin, and the resulting crosslinked nucleotide peptides were enriched using titanium dioxide beads. Crosslinks were identified from dda-PASEF data using OpenNuXL software. Spectral libraries were created for ribosome results, and dia-PASEF data were analyzed using DIA-NN. Collisional cross-sections of identified crosslinks were generally lower than those of peptides of the same m/z, most likely due to the more compact structures of the crosslinks. Additionally, it was found that mono- or dinucleotide additions consistently increased the collisional cross-sections (CCS) compared to corresponding linear peptides, with the presence of uridine monophosphate leading to a decrease in CCS of crosslinks. To conduct quantitative analysis, we used different UV exposure times and NM concentrations to model various crosslink concentrations. We quantified 39 UV crosslinks at all five irradiation time points ranging from 30 seconds to 10 minutes, compared to 112 crosslinks in the 10-minute treatment. In addition, we quantified 463 linear peptides at all time points. The relative intensity of crosslinks returned by DIA-NN followed a logarithmic function with saturation close to the 5-minute exposure point. Similar results were obtained for NM crosslinks. When using NM concentrations of 0.1, 1, 5, and 10 mM, we quantified significant numbers of crosslinks, with 223 species compared to 314 peptides at all four points. In both treatments, crosslinks exhibited high fitness and steep slopes in the linearized logarithmic regression curves, unlike peptides. This quantitative analysis of RNA-protein crosslinks using data-independent acquisition opens up new possibilities for system biology analysis in relevant biological conditions and models, such as viral infections and translation-targeting drug treatments.