Proteome wide interactomics analysis using MS-cleavable crosslinkers and the Orbitrap Astral Zoom mass spectrometer

Significance of the Topic

The study addresses the growing demand for proteome-wide mapping of protein structures and interaction networks. Crosslinking mass spectrometry (XL-MS) combined with MS-cleavable reagents has emerged as a powerful approach to capture both intra- and inter-protein contacts in complex systems. However low abundance of crosslinked peptides and lengthy acquisition times limit broader adoption. Integrating advanced mass analyzers with optimized fragmentation schemes promises to overcome these challenges and expand XL-MS applications in structural biology and interactomics.

Objectives and Overview of the Study

This work presents an end-to-end XL-MS workflow for the cleavable crosslinkers DSSO and DSBSO on the Orbitrap Astral Zoom mass spectrometer. The main goals were to optimize collision energy settings and scan modes to maximize identification rates, compare performance against conventional Orbitrap Astral acquisition, and evaluate throughput and sensitivity on E coli ribosome samples and a defined human protein mix.

Methodology and Instrumentation

Sample Preparation and Crosslinking

• E coli ribosomal proteins and a 64-protein human mix were buffer exchanged and crosslinked at 8 mg/ml with 100× molar excess of DSSO or DSBSO.
• Reactions were quenched, reduced, alkylated, and digested with trypsin overnight.
• DSBSO-crosslinked peptides underwent bio-orthogonal enrichment using DBCO magnetic beads.

Data Acquisition

• Liquid chromatography employed a Vanquish Neo UHPLC system with a 25 cm EASY-Spray column and gradients of 20 to 60 minutes.
• Mass spectrometry used the Orbitrap Astral Zoom MS with FAIMS Pro Duo interface. MS1 scans were acquired in the Orbitrap at 180 k resolution; MS2 scans utilized the Astral analyzer with stepped collision energy (SCE) methods.
• FAIMS compensation voltages of -48, -60, and -70 V were applied for enhanced selectivity.

Data Analysis

• Proteome Discoverer 3.2 with the XlinkX node performed an XL-open search against appropriate databases, using 10 ppm MS1 and 20 ppm MS2 tolerances.
• False discovery rates were set at 1% for both crosslink spectral matches and overall crosslinks.

Used Instrumentation

• Thermo Scientific Orbitrap Astral Zoom mass spectrometer
• Thermo Scientific FAIMS Pro Duo interface
• Thermo Scientific Vanquish Neo UHPLC system
• Thermo Scientific EASY-Spray HPLC column (75 µm × 25 cm)

Main Results and Discussion

Collision Energy Optimization

• Two-step SCE (25-32) provided on average 10% more crosslink identifications than three-step schemes.
• Top N acquisition outperformed top-speed approaches by ca. 5% in crosslink yield.
• Crosslinker-dependent fragmentation differences were observed, with DSBSO favoring a two-charge state method.

Enhanced Throughput

• The Astral Zoom MS achieved similar crosslink numbers in half the gradient length compared to Orbitrap-only DDA, demonstrating a twofold speed increase.
• Short 20-minute gradients delivered equivalent identifications to conventional 60-minute runs, tripling analytical throughput.

Improved Sensitivity and Spectrum Quality

• In a human-to-E coli spiking series, Astral Zoom MS detected 53% more crosslinks at low abundance (1 : 10 ratio) and extended detection down to 1 : 50 ratios.
• Astral MS2 spectra exhibited higher sequence ion coverage and XlinkX scores versus Orbitrap MS2, enhancing confidence in crosslink assignment.

Benefits and Practical Applications of the Method

The optimized workflow on the Astral Zoom MS delivers markedly higher inter-protein crosslink detection and improved confidence scores while reducing analysis time. This makes it well suited for large-scale interactome studies, structural modeling, and quality control in pharmaceutical and biotechnology research where fast, sensitive PPI mapping is critical.

Future Trends and Possibilities

Integration with real-time search and AI-driven algorithms can further accelerate XL-MS data processing. Coupling with ion mobility separations or deeper fractionation strategies may enhance coverage in highly complex proteomes. Advances in cleavable crosslinker chemistry and automated platforms will likely broaden accessibility and standardization of XL-MS in structural biology and systems proteomics.

Conclusion

This work demonstrates that the Orbitrap Astral Zoom mass spectrometer, combined with optimized stepped collision energies and FAIMS, significantly advances the speed, sensitivity, and coverage of MS-cleavable XL-MS workflows. The result is an efficient platform for proteome-wide characterization of protein interactions with high confidence and throughput.

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