Comprehensive and Robust Proteome Profiling using Online-2D NanoLC coupled to the Orbitrap Exploris 480 MS

Significance of the Topic

Comprehensive proteome profiling is essential for understanding biological systems, discovering biomarkers and advancing drug development. Conventional one-dimensional LC-MS/MS methods often lack the depth required to detect low-abundance proteins in complex samples. Integrating an online two-dimensional nanoLC approach with high-resolution Orbitrap MS addresses these limitations by enhancing separation efficiency, throughput and sensitivity.

Objectives and Overview

This study presents an automated online low-flow high-pH reversed-phase (RP) × low-pH RP two-dimensional nanoLC platform (online 2D-nanoLC) coupled to an Orbitrap Exploris 480 mass spectrometer. The goals are to simplify sample handling, improve orthogonality between separations, increase proteome coverage and demonstrate robustness in both standard (HeLa digest) and challenging (crude human serum) matrices.

Methodology and Data Analysis

• Sample Preparation: HeLa protein digest was reconstituted in 0.1% formic acid. Serum proteins underwent methanol precipitation and trypsin digestion without cysteine reduction or alkylation, in a 96-well plate format.
• Online 2D-nanoLC Workflow: First-dimension separation employed high-pH RP on a PepSwift monolithic capillary column. Fractions were captured alternately on two trap columns and eluted with staggered low-pH RP gradients on an EASY-Spray column for the second dimension, minimizing carry-over and maximizing MS utilization.
• Data Acquisition: The Orbitrap Exploris 480 operated in data-dependent acquisition (DDA) mode, enabling high resolution MS1 surveys and rapid MS/MS scans.
• Data Processing: Proteome Discoverer 2.2 searched against the UniProt Homo sapiens database with a precursor mass tolerance of 10 ppm, fragment tolerance of 0.02 Da, trypsin specificity, two missed cleavages, fixed carbamidomethylation and variable methionine oxidation/deamidation. Identifications were filtered at 1% FDR.

Instrumentation Used

• Thermo Scientific UltiMate 3000 RSLCnano system
• Thermo Scientific Orbitrap Exploris 480 mass spectrometer
• Thermo Scientific PepSwift monolithic capillary column (100 µm × 250 mm)
• Thermo Scientific EASY-Spray column (75 µm × 15 cm)
• Trap columns and HyperSep C18 cartridges (100 mg bed weight)

Key Results and Discussion

• Fraction Methods: Increasing from 2 to 8 fractions yielded a linear rise in peptide and protein identifications, achieving up to 88% MS duty cycle in the 8-fraction method.
• Orthogonality: Peptide hydrophobicity distributions confirmed effective separation in both dimensions, ensuring broad coverage and reduced sample overlap.
• HeLa Digest: Demonstrated consistent peptide distribution and high reproducibility across fractions, supporting method scalability.
• Serum Profiling: The 4-fraction approach increased protein group identifications by ~30% over 2 fractions and enabled profiling of 15 serum samples in ~57 hours, averaging 321 protein groups and 3,658 peptides per sample.

Benefits and Practical Applications

• Eliminates manual fraction collection and desalting, reducing sample loss and labor.
• Offers high throughput for large clinical cohorts with minimal carry-over.
• Compatible with diverse sample matrices, including crude serum.
• Provides an alternative to extended one-dimensional gradients for deep shotgun proteomics.

Future Trends and Perspectives

Further enhancements may include integration of isobaric labeling or ion-mobility separations, expansion to higher fraction numbers and coupling with quantitative workflows. Advances in software for real-time decision making and AI-driven method optimization are expected to further increase depth and throughput.

Conclusion

The online 2D-nanoLC coupled to an Orbitrap Exploris 480 delivers a robust, high-orthogonality workflow for deep proteome profiling. It combines automation, high sensitivity and efficient MS utilization, making it a powerful tool for both discovery and routine analysis in proteomics.

References

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