Breaking Barriers in Crosslinking Mass Spectrometry: Enhanced Throughput and Sensitivity with the Orbitrap Astral Mass Analyzer

The advancement of crosslinking mass spectrometry (CLMS) has significantly enhanced the ability to study protein-protein interactions and complex biological systems. This study evaluates the performance of the Orbitrap Astral and Eclipse mass spectrometers in CLMS workflows, focusing on the identification of low-abundance crosslinked peptides. The comparison employed consistent liquid chromatography setups and experimental conditions, using Cas9 crosslinked with PhoX and DSSO as quality control samples. Results demonstrated that the Astral analyzer outperformed the Eclipse, achieving over 40% more unique residue pairs (URP) due to its superior sensitivity and dynamic range, attributed to its multi-reflection time-of-flight analyzer and nearly lossless ion transmission. Additionally, the study revealed that single higher-energy collisional dissociation (HCD) fragmentation methods significantly outperformed stepped HCD methods on the Astral, while the Eclipse maintained similar performance across both approaches. Gradient optimization experiments further highlighted the impact of separation times on crosslink identifications, with longer gradients yielding higher identification rates. Collectively, this work underscores the importance of instrumentation choice, fragmentation strategies, and method optimization in maximizing CLMS performance for protein interaction studies.