Large Panel Targeted Proteomics for Low-amount Samples Using an Enhanced Nanoflow 6495D LC/TQ System

Significance of the Topic

Quantitative proteomics of extremely low sample amounts, including single cells, is critical for biomarker discovery, disease mechanism studies and precision medicine. Improving sensitivity, selectivity and throughput of targeted proteomics workflows enables reliable measurement of hundreds to thousands of proteins from minute biological specimens.

Objectives and Study Overview

The study aimed to develop and demonstrate a large-panel targeted proteomics workflow for low-amount samples using an enhanced nanoflow LC/TQ system. Key goals included maximizing detection sensitivity and precision without relying on stable isotope-labeled standards, and validating quantification performance down to single cell protein levels.

Methodology and Instrumentation

Instrumentation

• Evosep One nanoflow liquid chromatography system
• Newomics UniESI 2.0 ion source (modified for robustness)
• Agilent 6495D triple quadrupole mass spectrometer
• Agilent AssayMAP Bravo platform for automated sample preparation

Workflow

1. Prepare serial dilutions of human K562 cell proteome digest (31.25 pg to 64 ng on-column).
2. Generate a dynamic MRM method targeting 1,000 proteins (one peptide per protein, eight product ions each) in Skyline with the Agilent automation plugin.
3. Predict and update peptide retention times using endogenous high-abundance peptides; optimize collision energies and transition selection.
4. Acquire data on the enhanced Evosep–Newomics–6495D LC/TQ system with a 68-minute gradient at 200 nL/min and column temperature of 50 °C.
5. Process data in Skyline for peak integration, quality filtering (dotp, peak shape), and quantification.

Main Results and Discussion

RT Consistency and Method Accuracy

• Comparison with DDA on a high-resolution LC/QTOF showed 98.5% of 456 common peptides had matching retention times (R² = 0.9989).
• Increasing transitions per peptide from four to eight improved detection accuracy from 68% to nearly 99%.

Reproducibility and Sensitivity

• Method covered 1,000 proteins with a total of 7,942 dMRM transitions; median peak area CV was 7.5% (n=8).
• Median full width at half maximum (FWHM) of peptide peaks was 0.1 min with ~17 data points per peak.

Quantification at Single Cell Level

• Linear calibration curves for individual peptides (e.g., histone LLLPGELAK, Poly(rC)-binding protein 1) showed R² > 0.99 from 31.25 pg to 64 ng on-column.
• Consistent triplicate measurements at low levels demonstrated robust detection of hundreds of proteins at single-cell equivalent amounts.

Practical Benefits and Applications

The described workflow offers:

• High sensitivity enabling single-cell protein quantification.
• Economic method development without stable isotope-labeled standards.
• High selectivity through optimized dynamic MRM transitions.
• Enhanced throughput using automated sample processing and extended dMRM capabilities.

Future Trends and Potential Applications

Advances may include:

• Expansion to deeper proteome coverage beyond 1,000 proteins using refined spectral libraries.
• Integration of real-time retention time alignment and AI-driven transition optimization.
• Further miniaturization and multiplexing for high-throughput single-cell analyses.
• Clinical application for early disease biomarker validation in limited patient samples.

Conclusion

The enhanced nanoflow LC/TQ platform combining Evosep One, Newomics ion source and Agilent 6495D achieves highly sensitive, precise and high-throughput targeted proteomics for low-amount samples. The economic workflow, free of isotope standards, reliably quantifies up to 1,000 proteins at single-cell levels, opening new possibilities for biomarker discovery and applications in clinical and research settings.

Reference

1 A Rapid and Economical Workflow for Protein Biomarker Discovery Using Agilent 6495D Triple Quadrupole LC/MS System