Quantitative Targeted Proteomics of Mouse Plasma Protein Biomarkers by using Nano LC coupled to Triple Quadrupole Mass Spectrometer

Significance of the Topic

The quantification of plasma protein biomarkers is fundamental for disease diagnosis, targeted therapy and monitoring therapeutic response. Mouse plasma represents a complex proteome spanning ten orders of magnitude in concentration, posing significant analytical challenges. Highly sensitive, precise and high-throughput targeted proteomics methods are therefore essential to translate biomarker discovery into practical blood tests.

Objectives and Overview of the Study

This study evaluated a nano-flow LC coupled to triple quadrupole mass spectrometry (Evosep One with Agilent 6495C QQQ) to quantify 375 mouse plasma proteins in a single 44-minute analysis. Using a PeptiQuant Plus MRM kit and stable isotope labeled standards, the goal was to demonstrate rapid acquisition of 2,250 MRM transitions without loss of sensitivity or precision.

Methodology and Instrumentation

• Sample Preparation: BSA digest as surrogate matrix; reduction by DTT, alkylation with iodoacetamide, overnight tryptic digestion; spiking of light peptides and SIS.
• Evotip Cleanup: Conditioning, loading and washing steps according to manufacturer protocol.
• LC/MS Setup: Evosep One nano-flow LC (30 SPD, 44 min gradient) coupled to Agilent 6495C triple quadrupole MS in dynamic MRM mode.
• Data Analysis: Skyline and Agilent Quantitative Analysis software for peak integration and calibration.

Main Results and Discussion

• Quantification of 375 target peptides (plus 375 SIS) with 2,250 transitions in one run; minimum dwell time 1.2 ms.
• Lower limit of quantification (LLOQ) below 100 amol/µL for over 85% of peptides, representing >10-fold sensitivity improvement versus conventional UHPLC.
• Calibration curves spanned four orders of magnitude (38.6 amol/µL to 965 fmol/µL) with bias <±10% and CV <10%.
• Intra- and inter-day precision and accuracy of quality control samples remained within ±15% bias and CV <6% across all levels.

Benefits and Practical Applications

• High analytical sensitivity enables detection of low-abundance plasma proteins.
• Excellent precision and accuracy support reliable quantitation in QA/QC and biomarker validation workflows.
• High throughput (30 samples per day) and robustness facilitate large-scale proteomic studies in translational research.

Future Trends and Applications

The integrated nano-flow LC–QQQ platform may be expanded to larger targeted panels, multiplexed assays and clinical diagnostics. Advances in automation, microfluidics and data processing will further enhance throughput and reproducibility. Integration with multi-omics approaches could drive personalized medicine applications.

Conclusion

This work demonstrates that coupling the Evosep One nano-flow LC to a 6495C triple quadrupole MS enables rapid, sensitive and precise quantification of hundreds of mouse plasma protein biomarkers in a single analysis, meeting key requirements for translational proteomics.

References

• Bache N et al. A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-robust Proteomics. Mol Cell Proteomics 2018;17(11):2284–2296.