Capture scheduled retention time window shift in large scale of peptide quantitation using a modified Orbitrap hybrid mass spectrometer

Significance of the Topic

High-throughput targeted peptide quantitation in complex matrices such as plasma is central to biomarker discovery and clinical research. Scheduled PRM methods enhance throughput by focusing acquisition on predefined retention time windows, but shifts in chromatographic conditions can lead to missing data and reduced assay robustness. Implementing real-time adaptive retention time alignment directly on the mass spectrometer addresses these challenges and improves data completeness and quality.

Objectives and Study Overview

This study aimed to develop and evaluate a large-scale targeted PRM workflow capable of quantifying approximately 800 peptides in a 30-minute LC gradient. Key goals included integrating an adaptive retention time (RT) function on a Thermo Scientific Orbitrap Excedion Pro hybrid mass spectrometer, assessing its performance under varying conditions, and demonstrating improvements in peptide coverage, linearity, accuracy, and precision.

Methodology and Instrumentation

The workflow combined peptide standards and plasma digest with advanced LC-MS instrumentation and real-time RT adjustment:

• Sample Preparation: PQ500 peptide mix (Biognosys AG) spiked into Thermo Scientific Pierce human plasma digest, serially diluted from 100% to 0.1% concentration levels.
• Liquid Chromatography: Thermo Scientific Vanquish Neo UHPLC, EASY-Spray ES906 column, 30-minute gradient at 0.8 μl/min, mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in 80% acetonitrile).
• Mass Spectrometry: Orbitrap Excedion Pro hybrid MS operated in three sequential experiments – an Adaptive RT full-scan to generate a reference, a full MS scan, and a scheduled PRM assay with 0.6-minute windows for ~800 peptides.
• Data Analysis: Skyline software for retention time visualization, real-time alignment, and quantitative evaluation of linearity, accuracy, precision, and sensitivity.

Main Results and Discussion

Adaptive RT performance was tested under changes in column temperature, flow rate, and column replacement:

• Retention Time Alignment: Adaptive RT dynamically shifted scheduled windows to track peptides, capturing over 90% of targets across varied conditions, compared with 13.8–57.8% coverage without adaptive adjustment.
• Quantitative Performance: In a 30-minute run, 800 peptides achieved excellent linearity (94.6% with R2 ≥ 0.9; 98% with R2 ≥ 0.8), high accuracy (within ±20%), and precision (CV < 25% for >94.5% of peptides) at attomole sensitivity.
• Time Efficiency: Eliminated the need for manual rescheduling of RT windows, enabling consistent high-throughput analysis.

Benefits and Practical Applications

• Enhanced Data Completeness: Real-time RT adjustment reduces missing peptide signals caused by chromatographic variability.
• Streamlined Workflow: No manual method updates required when switching columns or changing conditions.
• High Throughput and Robustness: Supports large-panel targeted assays for biomarker validation, QA/QC, and industrial analytics.

Future Trends and Applications

Adaptive retention time alignment can be extended to data-independent acquisition (DIA) workflows and larger peptide panels. Integration with machine learning models could further predict and correct chromatographic shifts. Automation of reference generation and feedback-driven method optimization will support clinical deployments and real-time quality control.

Conclusion

Implementing adaptive RT on an Orbitrap hybrid mass spectrometer enables reliable, high-throughput quantitation of hundreds of peptides in fast LC gradients. This innovation enhances assay robustness by correcting retention time shifts in real time, delivering precise, accurate, and sensitive measurements without manual intervention.