An Easier, More Robust, and Faster Low- flow Quantitative Proteomics Approach

Significance of the Topic

Targeted proteomics using multiple reaction monitoring (MRM) has become a cornerstone in quantitative protein analysis, especially when moving from broad discovery workflows to precise, hypothesis‐driven assays. Challenges such as low protein abundance and high sample complexity drive ongoing innovation in chromatography and mass spectrometry. Integrating fast, low‐flow liquid chromatography systems with modern triple quadrupole instruments promises improved sensitivity, throughput, and robustness, meeting the stringent demands of biomarker validation, clinical research, and quality control in complex matrices.

Objectives and Overview of the Study

This work evaluated a combined platform comprising a high‐performance low‐flow HPLC system and a triple quadrupole mass spectrometer for rapid, precise, and robust MRM‐based quantitation of peptides in plasma. Key aims included:

• Reducing analysis cycle time while maintaining or improving sensitivity and linear dynamic range.
• Testing the reproducibility and lower limit of quantitation (LLOQ) under accelerated acquisition conditions.
• Demonstrating the platform’s suitability for high‐throughput targeted proteomics with minimal sample consumption.

Methodology

Human plasma was tryptically digested and spiked with stable isotope‐labeled standard (SIS) peptides from a commercial biomarker kit. Peptide mixtures were separated using three predefined sample‐per‐day (SPD) gradients—60, 200, and 300 SPD—on a low‐flow HPLC system, achieving flow rates of 1–4 µL/min. MRM transitions were optimized in Skyline, targeting up to 250 peptide transitions per run. Quantitation was performed on a triple quadrupole instrument, and data were processed with quantitative and qualitative analysis software.

Instrumentation

• Low‐flow chromatography: Evosep One HPLC with standardized gradient cartridges (60, 200, 300 SPD).
• Mass spectrometer: Agilent 6495C triple quadrupole LC/TQ.
• Ion source: Agilent nano ESI emitter and capillary interface.
• Software: Skyline for method development; Agilent Quantitative Analysis and Qualitative Analysis for data processing.

Main Results and Discussion

Using the 60 SPD (21 min) method, 125 peptides were quantified with an LLOQ defined by ≤15% CV, despite dwell times as low as 7 ms. Peak widths were typically <3.6 s, and all transitions eluted by 18 min. Shortening dwell times to 5 ms still yielded >90% of peptides with <15% CV across ten replicates. Lower limit of quantitation analysis showed that 50% of peptides had LLOQs under 100 amol on column, representing a substantial sensitivity gain compared to the standard kit.

High‐throughput 200 and 300 SPD methods (5.6 and 3.2 min) were tested on a subset of 20 peptides. Even at these accelerated rates, 30% of targets achieved LLOQs below 50 amol, and 55% below 500 amol, demonstrating that modest target lists benefit from ultra‐fast chromatography without a large drop in sensitivity.

System robustness was highlighted by over 1,500 injections with no loss of performance or increased carryover, facilitated by disposable trap columns and simple finger‐tight fittings.

Benefits and Practical Applications of the Method

• High analytical sensitivity and extended linear dynamic range down to low‐amol levels.
• Fast sample throughput with cycle times under 25 min (60 SPD) and under 6 min for focused assays.
• Robust operation with minimal carryover and maintenance requirements.
• Simplified data processing using established software tools.

Future Trends and Potential Applications

Continued development may focus on integrating microfluidic-based gradient formation, even higher acquisition speeds, and automated sample handling to further boost throughput. Expansion into clinical diagnostics, large‐scale biomarker validation studies, and real‐time therapeutic drug monitoring are promising areas. In addition, coupling with ion mobility or high-resolution mass analyzers could enhance selectivity and multiplexing capabilities.

Conclusion

This study demonstrates that coupling a low‐flow HPLC system with a modern triple quadrupole instrument enables fast, sensitive, and highly reproducible MRM assays. The approach supports high-throughput targeted proteomics workflows, making it well suited for biomarker validation and other quantitative applications where speed and robustness are critical.

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.