A Fully Integrated Qual/Quan Solution from a Single Injection Using Standardized Sample Preparation and Chromatography with a Benchtop MRT Mass Spectrometer

Integrated Single-Injection Qual/Quan LC–MS Workflow Using the Xevo MRT P10

Significance of the Topic

Modern proteomic and routine LC–MS laboratories must balance broad qualitative characterization with precise quantitative measurement while maximizing throughput and reproducibility. Combining discovery-oriented data-independent acquisition with targeted multiple-reaction monitoring in a single injection reduces instrument time, sample consumption, and between-run variability, making high-throughput and longitudinal studies more practical and reliable.

Objectives and Study Overview

This application note evaluated a mixed-mode LC–MS workflow that merges untargeted DIA (MSe) and scheduled Tof MRM acquisition in a single 24-minute run. The study aimed to demonstrate that standardized automated sample preparation and Evosep chromatography, coupled with fast multi-reflecting TOF acquisition on a benchtop Xevo MRT P10, can: deliver robust targeted quantification, retain comprehensive qualitative coverage, and maintain performance across extended injection sequences.

Methodology and Used Instrumentation

Sample and preparation

• Proteolytic sample: equimolar tryptic digest of four proteins spiked into a complex Escherichia coli tryptic digest to emulate a realistic matrix.
• Automated sample handling: Evotips were prepared using the Andrew+ automated pipetting robot for consistent cleanup, desalting, and sample loading.

Chromatography

• Evosep Eno System operated with a standardized 60 samples-per-day (SPD) method (24-minute total cycle including gradient, wash, equilibration).
• Disposable trap-based pre-formed gradients provide reproducible peptide retention and minimize between-run overhead.

Mass spectrometry and acquisition strategy

• Xevo MRT P10 Mass Spectrometer configured for mixed-mode acquisition: DIA (MSe) for untargeted coverage plus scheduled Tof MRM for targeted quantification.
• Target panel: ten predefined peptides monitored using >30 Tof MRM transitions within the same injection.
• Fast multi-reflecting TOF analyzer supports high acquisition speed sufficient for concurrent DIA and MRM without lengthening chromatographic cycles.

Data processing

• Targeted MRM processing: MS Quan Application within waters_connect for automated quantitation, batch review and consistent evaluation across large sample sets.
• Untargeted DIA processing: conversion to mzML and analysis with DIA-NN for protein and peptide identification; targeted and untargeted data paths processed independently to retain workflow flexibility.

Main Results and Discussion

• Single-run Qual/Quan: The mixed-mode method captured both DIA and scheduled Tof MRM data within each 24-minute LC cycle, preserving peak definition and quantitative integration even when acquisition events overlapped.
• Chromatographic stability: The Evosep 60 SPD method yielded highly reproducible retention times, with peptide RTs maintained within ±2 seconds of the mean across repeated injections—critical for scheduled targeted acquisition.
• Quantitative robustness: Scheduled Tof MRM responses remained stable and reproducible across 300 consecutive injections, supporting long-term studies and high-throughput operation.
• Qualitative coverage: Concurrent DIA acquisition provided consistent peptide and protein identifications throughout the sequence; processed samples showed peptide IDs within ~10% of the mean for regularly sampled injections, indicating minimal loss of qualitative performance.
• Operational advantages: Automated Evotip loading reduced manual variability, contributing to stable responses and simplifying sample throughput.

Key implications of these findings

• The multi-reflecting TOF’s acquisition speed enables simultaneous DIA and MRM without compromising chromatographic performance, eliminating the need for separate Qual and Quan injections.
• Standardized chromatography and automation are essential enablers for scheduled targeted acquisition at scale, reducing calibration overhead and increasing reproducibility across large batches.

Benefits and Practical Applications

• Reduced sample consumption and instrument time by consolidating discovery and targeted assays into one injection.
• Improved throughput suitable for routine laboratories, longitudinal studies, and biomarker verification workflows.
• Robust quantitative performance with accurate-mass confirmation (Tof MRM) combined with broad qualitative context from DIA supports confident decision-making in research and QC environments.
• Automated, standardized front-end processing lowers operator-to-operator variability and simplifies method transfer between labs.

Future Trends and Potential Uses

• Scalability: expansion of targeted panels and multiplexing strategies to broaden clinical and industrial applications.
• Adaptive acquisition: real-time or scheduled adaptive methods that leverage initial DIA information to refine targeted windows dynamically.
• Deeper automation: tighter integration of robotics, sample tracking, and laboratory information management systems (LIMS) for regulated environments.
• AI-driven processing: machine learning tools to streamline identification, transition selection, interference detection, and automated QC across large cohorts.
• Standardization and benchmarking: development of community standards for single-injection Qual/Quan assays to support regulatory acceptance and inter-lab comparability.

Conclusion

The mixed-mode LC–MS workflow implemented on the Xevo MRT P10 demonstrates that concurrent DIA and scheduled Tof MRM acquisition can produce comprehensive qualitative coverage and robust targeted quantification from a single injection. When combined with automated Evotip preparation and standardized Evosep chromatography, the approach enhances throughput, reduces sample consumption, and maintains reproducibility across extended sequences—making it a practical solution for routine and high-throughput proteomic analyses.

Used instrumentation

• Xevo MRT P10 Mass Spectrometer (multi-reflecting TOF)
• Evosep Eno System (Evosep 60 SPD standardized method)
• Evotips for trap-based sample loading
• Andrew+ automated pipetting robot for Evotip preparation
• Software: waters_connect with MS Quan Application (MRM processing), mzML conversion and DIA-NN (DIA processing)

References

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