Large-Panel Targeted Proteomics with Single-Cell-Equivalent Sensitivity Using Nanoflow 6495D LC/TQ

Significance of the Topic

Quantitative proteomics at single-cell sensitivity is critical for understanding cellular heterogeneity in fields such as cancer and immunology where sample amounts are extremely limited. The ability to detect and quantify subtle differences in protein expression and post translational modifications can reveal key biological insights masked in bulk analyses.

Study Objectives and Overview

This application demonstrates the integration of nanoflow liquid chromatography and triple quadrupole mass spectrometry to achieve large panel targeted proteomics with single-cell equivalent sensitivity. A dynamic multiple reaction monitoring assay was developed to quantify 1 000 proteins in human K562 cell lysate across sample loads from 31.25 pg to 64 ng on column.

Methodology

Sample preparation involved automated loading of a K562 digest on Evotips using the AssayMAP Bravo platform and generating a serial dilution series. Skyline software guided targeted method development including spectral library selection peptide filtering retention time prediction and transition optimization. A single dMRM method containing 7 942 transitions was exported for data acquisition.

Instrumentation

• Evosep One nanoflow LC system
• Newomics UniESI ion source for nanospray
• Agilent 6495D triple quadrupole LC MS
• Agilent AssayMAP Bravo sample preparation platform
• IonOpticks Aurora Elite XT C18 UHPLC column

Main Results and Discussion

The optimized platform supports up to 500 concurrent transitions with a cycle time of 1 400 ms. Reproducibility was high with a median peak area CV of 7.5 and 96.2 of peptides showing CV below 20. Retention times matched data dependent acquisition on high resolution MS with 98.5 consistency. Quantification was linear across five orders of magnitude with 57.8 of proteins showing R2 above 0.99. Notably 10 of targets were detectable at 31.25 pg on column approximating single cell protein content.

Benefits and Practical Applications

• High sensitivity and specificity enabling low level biomarker detection
• Cost effective workflow without the need for synthetic labeled standards
• Scalable assay quantifying 1 000 proteins in a single run
• High throughput automation for clinical translational and QA QC labs

Future Trends and Opportunities

The approach can be extended to diverse cell types clinical specimens and post translational modification studies. Continued software advances will streamline method development and retention time prediction. Integration with emerging ion sources and higher multiplexing will further expand panel size and throughput.

Conclusion

This study presents a robust integrated nanoflow LC TQ workflow achieving large panel targeted proteomics at single cell equivalent sensitivity. The combination of automated sample handling with a 1 000 protein dMRM assay on the Agilent 6495D system delivers high precision sensitivity and throughput for applications in biomarker discovery and translational research.

References

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