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Introduction Immunopeptidomics plays a crucial role in identifying targets for immunotherapy and vaccine development. Because immunopeptides are generated from their parent proteins in an unpredictable manner, rather than being able to use known digestion rules, every possible protein subsequence needs to be considered. This leads to an inflation of the search space and results in a low spectrum identification rate. Rescoring is a powerful enhancement of standard database searching that boosts the spectrum identification sensitivity and accuracy by unlocking the intensity dimension of MS/MS spectra with peptide fragment intensity predictions. The high sensitivity of a timsTOF instrument makes it ideal for detecting immunopeptides that are present at relatively low abundances. To improve the identification rate and the reliability of immunopeptidomics experiments performed using timsTOF instruments, we developed an optimized fragment ion intensity prediction model based on Prosit. Methods We analyzed over 300,000 synthesized non-tryptic peptides from the ProteomeTools project on a TimsTOF-Pro (Bruker, Bremen). The spectra were searched using MaxQuant (version 2.1.2.0) at 1% PSM-level FDR. The 277,781 obtained PSMs (93,227 non-tryptic measured in this study and 184,554 previously published tryptic peptide PSMs) were split into training (80%), validation (10%), and test (10%) sets. The training set was used to fine-tune the existing Prosit fragment intensity prediction model, the validation set to control for overfitting with early stopping, and the test set to evaluate the model. We reprocessed immunopeptidomics timsTOF data from a recent study using MaxQuant (version 2.0.3.1) and rescored all proposed PSMs by integrating the fragment intensity predictions. Results Comparison of the previously published and the here developed Prosit models showed a substantially improved normalized spectral contrast angle (SA) between predicted and experimental spectra for non-tryptic peptides (SA ≥ 0.9 for 2.4% vs 26.3% of spectra, respectively) and for tryptic peptides (SA ≥ 0.9 for 0.2% vs 42.1%). To evaluate whether rescoring with our model is able to increase the identification rate we reprocessed public HLA Class I and Class II immunopeptidome data. Similarly to what was observed previously on Orbitrap instruments, incorporating our model into the database matching process increased the spectrum identification rate of immunopeptides measured on a timsTOF. Compared to MaxQuant, we identified 3.0-fold more HLA class I peptides and 1.7-fold more HLA class II peptides after rescoring. To evaluate the clinical relevance of rescoring, we will look for peptides exclusively presented by tumors. We hypothesize that rescoring results in an increased reliability and identification rate of neoepitopes.

BackgroundRNA binding proteins regulate gene expression at the post-transcriptional level by controlling the fate of RNA, in processes such as mRNA localization, translation, splicing and stability. The annotation of RNA binding proteins is mainly based on the well-known RNA binding domains and motifs. However, novel RNA binding proteins without such conventional domains have been identified in different species using in vivo RNA interactome capture. To find support for novel conserved RNA binding proteins in plants, we applied an optimized RNA interactome capture to the monocot model Brachypodium distachyon.ResultsWe provide experimental evidence for 203 RNA binding proteins isolated from Brachypodium shoot tissue and leaf mesophyll protoplasts, and grouped these into classic RNA binding proteins with recognizable RNA binding domains and motifs, and candidate RNA binding proteins without such domains. Compared to RNA binding proteins captured in Arabidopsis thaliana, candidate RNA binding proteins involved in carbon fixation and carbon metabolic pathways are highly conserved. We tried to validate the RNA binding proteins captured in this research through a silica-based method, but this method appears not efficient for plants. This may indicate that optimized methods to validate high throughout RNA binding proteome are required for plants.ConclusionsOur results provide classic and candidate RNA binding proteins in Brachypodium distachyon and conserved RNA binding proteins in flowering plants. Future functional characterization should point out what the significance of RNA binding is for the function of these proteins.