Breaking barriers in crosslinking mass spectrometry with enhanced throughput and sensitivity using Orbitrap Astral.

ABSTRACT: Crosslinking mass spectrometry is an essential tool for probing protein-protein interactions and structural organization. We here compare Orbitrap Astral and Orbitrap Eclipse instruments using Cas9 crosslinked with PhoX and DSSO under standardized chromatographic and acquisition conditions. The Astral identifies over 40% more unique residue pairs, largely due to increased MS1 sensitivity and efficient detection of low-abundance precursors. Implementation of high-field asymmetric ion mobility spectrometry further increases identifications by 30% through improved precursor filtering. On the Astral, single higher-energy collisional dissociation consistently outperforms stepped fragmentation, particularly at low sample amounts, whereas the Eclipse shows minimal dependence on fragmentation stra

SUBMITTER: Muller F 

PROVIDER: S-EPMC12603054 | biostudies-literature | 2025 Nov

REPOSITORIES: biostudies-literature