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

ABSTRACT: Crosslinking mass spectrometry (XL-MS) has become a key technique for investigating protein-protein interactions and the architecture of biological systems. This study compares the Orbitrap Astral and Eclipse mass spectrometers in XL-MS workflows, emphasizing their performance in detecting low-abundance crosslinked peptides. Using Cas9 crosslinked with PhoX and DSSO, the Astral demonstrated over 40% higher crosslink identifications due to its superior ion transmission and sensitivity, enabled by its multi-reflection time-of-flight analyzer. Single higher-energy collisional dissociation (HCD) fragmentation was more effective than stepped HCD on the Astral, while the Eclipse showed similar results for both methods. Gradient optimization revealed that extended separation times improved crosslink identifications. These findings highlight the critical role of instrumentation and workflow optimization in advancing XL-MS for structural proteomics.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Fränze Müller

LAB HEAD: Karl Mechtler

PROVIDER: PXD059096 | Pride | 2025-12-15

REPOSITORIES: Pride

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			Action	DRS
	20240609_Astral_Neo1_Mueller_MS_TechHub_IMP_THIDDIAXL001_Cas9_PhoX_CV48_100ng_FAIMS_001.msf	Msf
	20240609_Astral_Neo1_Mueller_MS_TechHub_IMP_THIDDIAXL001_Cas9_PhoX_CV48_100ng_FAIMS_001.raw	Raw
	20240609_Astral_Neo1_Mueller_MS_TechHub_IMP_THIDDIAXL001_Cas9_PhoX_CV48_100ng_FAIMS_001_1.msf	Msf
	20240609_Astral_Neo1_Mueller_MS_TechHub_IMP_THIDDIAXL001_Cas9_PhoX_CV55_100ng_FAIMS_001.msf	Msf
	20240609_Astral_Neo1_Mueller_MS_TechHub_IMP_THIDDIAXL001_Cas9_PhoX_CV55_100ng_FAIMS_001.raw	Raw

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