Protein–RNA crosslinking combined with MS becomes quantitative at the peptide and amino-acid level

Mass Spectrometry can resolve UV- and chemically induced protein-RNA crosslinking at the peptide and amino-acid level. Our search engine, NuXL, improves the identification of protein-RNA UV/chemical crosslinks at amino-acid resolution, irrespective of sample complexity (isolated complexes or cellular entities). However, the quantification of crosslinks remains a challenge for the sensitive and precise detection of the abundance changes among peptides crosslinked under different cellular conditions or states. UV crosslinks various amino acids to mainly uracil, whereas chemical crosslinking connects specific amino acids to guanosine and adenosine. Accurate MS-based quantification requires monitoring of the same crosslinked species derived from different states, and this can be hampered by changes in crosslinking patterns. Here, we explored a label-free quantification (LFQ) approach to analyze UV and chemical crosslinks using the NuXL node in Thermo Scientific™ Proteome Discoverer™ software.

We used the E. coli ribosome to monitor UV- or chemically-induced protein-RNA crosslinking quantitatively by MS. Different amounts of E. coli ribosomes were exposed to UV or chemical crosslinkers and enriched using different reagents. After the sample preparation, samples were separated using a Thermo Scientific™ Vanquish™ Neo LC system with a 60 min gradient using an IonOpticks Aurora Ultimate™ (75 um x 25 cm) column. The yield of crosslinked peptide-RNA oligonucleotides was measured by their intensities in MS1 (label-free) using a Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer.

The NuXL node in Protoeome Discoverer 3.1 software, based on the OpenMS software package, was used for analyzing XLMS data from UV or chemically crosslinked protein–RNA samples. In this study, we extend the use of this tool to include the quantitative analysis of crosslinks. We investigated the feasibility of quantitatively analyzing UV or chemical crosslinking with different amounts of E. coli ribosomes. Although quantitative label-free MS is generally straightforward, it is challenged here by a final TiO2 enrichment step before LC-MS analysis during sample-processing of the crosslinked peptide-RNA oligonucleotide species. We evaluated and annotated the quantitative label-free results with respect to (i) the background of non-crosslinked species in the input, i.e., before the final enrichment step; (ii) non-bound peptides after TiO2 enrichment, and (iii) residual non-crosslinked peptide species in the TiO2-enriched sample, in order to optimize UV or chemical crosslinking sample preparation and enrichment protocols. We were able to identify and quantify more than 360 UV and 1400 chemically crosslinked peptides for over 300 unique peptide sequences. In conclusion, label-free quantification of crosslinked peptide-RNA oligonucleotides can be achieved accurately and reproducibly using the NuXL node embedded in Proteome Discoverer 3.1 software for complex samples.