van den Toorn, H. W. P. | bioRxiv Science

bioRxiv2026

Protein Language Model Decoys for Target Decoy Competition in Proteomics: Quality Assessment and Benchmarks

Large-scale proteomics relies heavily on target-decoy competition for false discovery rate estimation in peptide identification, and the performance of this strategy depends strongly on the design of the decoy database. Classical generators such as reversal and shuffling remain widely used. Here, we introduce the first protein language model-based (PLM) decoy generation for peptide identification and benchmark it against classical strategies. We evaluate these approaches using three complementary quality-control layers: sequence-based separability, search-engine-agnostic spectral-space diagnostics, and end-to-end mass spectrometry benchmarks, including pipelines with rescoring. Across these analyses, PLM-based decoys are harder for sequence-only neural networks to distinguish than most classical generators, suggesting fewer obvious sequence-level artifacts. However, this signal is only weakly informative for search performance. Spectral diagnostics further show that short peptides occupy a particularly crowded target-decoy space and are therefore especially prone to local collisions across all generators. In full search pipelines, reverse decoys remain a strong baseline, and current PLM-based generators do not yet provide a clear overall advantage. We therefore view PLM-based decoys not as universal replacements for reverse decoys, but as tunable tools for benchmarking, diagnostics, stress testing, and future adaptive decoy optimization, with increasing value as search models become more expressive.

bioinformatics↗

bioRxiv2020

Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

Ion mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a time-of-flight mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking mass spectrometry (XL-MS). Cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent - termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 [A]2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50 - 70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

biochemistry↗