Featuring Thermo Scientific Orbitrap Exploris 480 Mass Spectrometers

Significance of the topic

Mass spectrometry–based proteomics drives modern life-science research by enabling highly sensitive, quantitative and high-throughput analysis of proteins and their modifications. The introduction of compact quadrupole-Orbitrap instruments such as the Thermo Scientific Orbitrap Exploris 480, often coupled with front-end devices (FAIMS, nano-LC) and advanced acquisition methods (DIA, PRM, TMT multiplexing), has transformed applications across cell biology, clinical biomarker discovery, microbial physiology and structural proteomics.

Objectives and study overview

This collection of application notes, white papers and research articles illustrates how DIA-based workflows on the Orbitrap Exploris 480 extend proteome coverage, improve quantitation precision and accelerate sample throughput. Studies range from rapid serum-sample preparation to in-depth phosphoproteome mapping, from host–pathogen interaction profiling to single-shot deep proteome analyses. They showcase real-world implementations in areas such as infectious disease (SARS-CoV-2), oncology, stem-cell biology, metabolic regulation and biomarker discovery for organ-specific injury.

Methodology and instrumentation used

Key methodological elements include:

• Data-independent acquisition (DIA) on Orbitrap Exploris 480 for consistent quantitation of thousands of proteins in single runs
• High-field asymmetric waveform ion mobility spectrometry (FAIMS) for enhanced proteome depth during short LC gradients
• Multiplexed TMT labeling combined with real-time search and parallel reaction monitoring (PRM) for targeted validation
• Optimized nano-LC setups (high-pH/low-pH 2D and online 2D) for deep coverage in single-shot and multi-shot experiments
• Customizable rapid-digestion and enrichment protocols for biofluids, extracellular vesicles and archived tissue samples

Main results and discussion

Across diverse sample types, DIA on Exploris 480 consistently delivered:

• Identification of >10 000 proteins in single-shot runs through ion mobility-assisted acquisition
• Quantitative precision (<10% CV) in complex clinical cohorts (serum, plasma, cerebrospinal fluid, urine)
• High phosphosite coverage (>20 000 phosphopeptides) enabling pathway analysis of insulin signaling, viral infection and cell-cycle control
• Robust biomarker panels for liver disease, ovarian and lung cancer, acute pancreatitis, neurological disorders and inflammatory syndromes
• Resolution of subcellular proteome and phosphoproteome dynamics at organelle-level spatial proteomics

Benefits and practical applications of the method

These workflows demonstrate practical advantages for end users:

• Streamlined sample preparation compatible with clinical throughput
• Automated acquisition and data processing pipelines for reproducible results
• Flexible quantitation strategies from discovery (DIA-MS) to validation (PRM, targeted DIA)
• Compatibility with archival and low-input materials, including FFPE tissues, dried biofluids and low-cell-number samples
• Direct applicability to biomarker discovery, drug mode-of-action studies, functional proteomics and precision medicine

Future trends and possibilities

Ongoing developments will further extend capabilities:

• Integration of single-cell proteomics with ultra-low-flow LC and ion mobility for cellular heterogeneity studies
• AI-driven real-time acquisition methods that optimize sampling of low-abundance peptides
• Improved microfluidic front ends for complete on-chip sample handling and cleanup
• Multi-omics integration combining proteome, metabolome and transcriptome in single workflows
• Cloud-based platforms for standardized data sharing, batch correction and biomarker model building

Conclusion

The ensemble of DIA-based applications on the Orbitrap Exploris 480 highlights a versatile, high-performance platform for deep, quantitative and reproducible proteomics across basic research and clinical contexts. By combining optimized hardware, novel acquisition schemes and robust bioinformatics, it addresses challenges in throughput, sensitivity and data consistency, paving the way for routine application in biomarker discovery and systems biology.

References

A selection of representative studies from the Thermo Scientific Orbitrap Exploris 480 application series including:

1. Kim M. et al. Mol. Cell. Proteomics (2020) 19(4):716–729.
2. Bouhaddou M. et al. Cell (2020) 182(1–28).
3. Shen F. et al. Anal. Chem. (2021) 93(3):1578–1585.
4. Nakamura R. et al. Int. J. Mol. Sci. (2021) 22:2565.
5. Stukalov A. et al. Nature (2021).
6. Mc Ardle A. et al. Clin. Chem. (2021).
7. Ctortecka C. et al. Mol. Cell. Proteomics (2021).
8. Wojtkiewicz M. et al. J. Mol. Cell. Cardiol. (2022) 162:20–31.
9. McCoin C.S. et al. Mol. Omics (2022).
10. Needham E.J. et al. Nat. Biotechnol. (2021).