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.### Competing Interest StatementOliver Raether and Markus Lubeck are employees of Bruker Daltonik GmbH, manufacturer of the timsTOF Pro, and thus disclose competing financial interests.

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