Thermo Scientific™ Orbitrap Exploris™ 240 mass spectrometer - Sensitivity–leading level sensitivity for the analysis of limited and precious samples

Significance of the topic

In proteomics research, sensitivity plays a pivotal role when dealing with limited or low-abundance samples. High-resolution and accurate-mass detection enable identification of minor protein species, facilitating insights into rare cells, early disease markers, and subtle biological processes.

Objectives and overview of the study

This study evaluates the performance of the Orbitrap Exploris 240 mass spectrometer with the FAIMS Pro interface for shotgun proteomics analyses at sample loads ranging from 10 ng to 200 ng. It includes a reproducibility assessment through five replicate injections of a 10 ng HeLa digest spiked with calibration peptides.

Methodology and used instrumentation

• Sample preparation: 10 ng HeLa Protein Digest Standard and 10 fmol Peptide Retention Time Calibration Mixture.
• Liquid chromatography: IonOpticks Aurora UHPLC column (250 mm × 75 µm, 1.6 µm) at 300 nL/min with a 90 min gradient (3–95% acetonitrile in 0.1% formic acid).
• Mass spectrometry: Orbitrap Exploris 240 with FAIMS Pro interface (compensation voltages −50 V and −70 V), employing data-dependent acquisition and high-resolution accurate-mass detection.
• Software: Thermo Scientific Proteome Discoverer version 2.4 with 1% PSM false discovery rate.

Main results and discussion

• At 10 ng load, over 5,300 peptide groups and 1,600 protein groups were identified with high confidence.
• Increasing sample load to 200 ng yielded proportional gains in identifications, demonstrating linear performance scaling.
• Five replicate injections of the 10 ng sample showed excellent reproducibility in retention time and precursor detection, including low-abundance peptides dominated by calibration standards.
• The FAIMS Pro interface enhanced selectivity by reducing background ions, enabling consistent detection of rare peptide precursors.

Benefits and practical applications

• Enables deep proteome coverage from minimal sample amounts, crucial for rare or precious specimens such as single cells and circulating tumor cells.
• High reproducibility supports quantitative studies and longitudinal experiments in clinical and biological research.
• Seamless integration with standard LC–MS workflows facilitates adoption in research and quality control laboratories.

Future trends and opportunities

• Advancements in nanoLC and ion mobility to further enhance depth and throughput for single-cell proteomics.
• Integration of real-time data analysis and machine learning for dynamic acquisition and method optimization.
• Expansion into clinical biomarker discovery and low-input diagnostic applications.

Conclusion

The Orbitrap Exploris 240 mass spectrometer with FAIMS Pro interface delivers best-in-class sensitivity and reproducibility for low-input shotgun proteomics. It supports robust identification and quantification of proteins from challenging samples, offering a versatile platform for cutting-edge biological and clinical research.

References

1. Cong Y, Liang Y, Motamedchaboki K, Huguet R, Truong T, Zhao R, Shen Y, Lopez-Ferrer D, Zhu Y, Kelly RT. Improved Single-Cell Proteome Coverage Using Narrow-Bore Packed NanoLC Columns and Ultrasensitive Mass Spectrometry. Anal. Chem. 2020, 92, 2665–2671.