An in-depth and high throughput plasma proteomics workflow powered by Orbitrap Exploris 480 mass spectrometer using multi nanoparticle-based workflow

Significance of the Topic

Plasma proteomics is a critical approach for identifying biomarkers of disease and understanding physiological states. Achieving both high throughput and deep coverage in plasma analysis poses substantial challenges due to the wide dynamic range of protein concentrations. The combination of nanoparticle-based enrichment with state-of-the-art mass spectrometry addresses these challenges, enabling large-scale studies without sacrificing sensitivity or reproducibility.

Study Objectives and Overview

The study presents two complementary workflows on the Orbitrap Exploris 480 mass spectrometer: a high-throughput method optimized for speed and robustness, and a Max-ID method designed for maximal protein identification. Both workflows employ Seer’s Proteograph XT Assay with multi-nanoparticle (NP) enrichment to achieve unbiased and in-depth plasma proteome analysis at scale.

Methodology and Instrumentation

Samples and Enrichment

• Pooled healthy control human plasma was processed using an automated SP100 system and the Proteograph XT Assay Kit.
• Two distinct nanoparticle chemistries were used per sample to form reproducible protein coronas, capturing high-affinity proteins and reducing abundant proteins interference.
• Protein coronas underwent reduction, alkylation, and on-NP digestion with Trypsin/Lys-C in a one-pot reaction.

LC-MS Parameters

• High-Throughput Workflow:
  
  • Vanquish Neo UHPLC with Fast Loading and Equilibration.
  • Easy-Spray PepMap Column (2 µm C18, 150 µm × 15 cm), trap-and-elute format at 1.5 µL/min.
  • Two 24-minute injections per sample (48 min total).
• Max-ID Workflow:
  
  • Direct injection on Vanquish Neo UHPLC.
  • Easy-Spray PepMap Neo Column (2 µm C18, 75 µm × 75 cm) at 0.25 µL/min.
  • Single 102-minute gradient injection.

MS Settings

• Orbitrap Exploris 480 with MS1 resolution 60,000 and MS2 resolution 15,000.
• AGC targets of 300% (MS1) and 800% (MS2) for high-throughput; 300% and 1000% for Max-ID.
• Isolation window of 4 Da; MS2 fill times auto (high-throughput) or 28 ms (Max-ID).

Data Analysis

• Primary analysis with Proteograph Analysis Suite (PAS) using match-between-runs (MBR).
• Library-free searches conducted in Spectronaut 18, DIA-NN, and Proteome Discoverer 3.1 with CHIMERYS at 1% FDR.

Main Results and Discussion

The high-throughput workflow identified over 3,000 protein groups and more than 20,000 peptides per sample, achieving 95% data completeness and 87.2% of proteins with CV below 20%. The Max-ID workflow extended coverage to over 3,800 protein groups and 27,000 peptides, with 99% completeness and 89.2% of proteins meeting CV <20%. Both methods demonstrated a dynamic range comparable to or exceeding the human plasma proteome project index. Cross-platform library-free searches confirmed robust quantitation and reproducibility across software tools.

Benefits and Practical Applications

These workflows enable large-cohort plasma studies with:

• High throughput for clinical and epidemiological applications.
• Deep proteome coverage for biomarker discovery.
• Robust automation and reproducibility via nanoparticle-based enrichment.
• Compatibility with multiple data analysis platforms.

Future Trends and Applications

Ongoing developments may include:

• Integration of artificial intelligence for automated data interpretation.
• Expansion to post-translational modification profiling in plasma.
• Further miniaturization and multiplexing to increase throughput.
• Application to personalized medicine and real-time clinical diagnostics.

Conclusion

The combined nanoparticle enrichment and Orbitrap Exploris 480 workflows deliver both rapid and in-depth plasma proteomics with high reproducibility. These methods establish a versatile platform for large-scale biomarker studies and clinical research.

References

• No literature references were listed in the source document.