Implementation of MS1 based real–time retention time adjustment on a hybrid Orbitrap mass spectrometer for directed and targeted proteomics

Significance of the Topic

The variability in chromatographic retention times due to column aging and gradient fluctuations impairs reproducibility and quantification in large‐scale targeted proteomics studies. Real‐time retention time alignment based on MS1 data offers a way to maintain narrow scheduling windows, increase target coverage and avoid frequent spiking of standard mixtures.

Aims and Study Overview

This work implements and validates a software and acquisition workflow for real‐time retention time adjustment on a modified Thermo Scientific Orbitrap Exploris 480 mass spectrometer. The method uses intermittent MS1 scans for cross‐correlation against a reference experiment to dynamically correct retention time shifts during data acquisition.

Methodology and Instrumentation

The procedure involves two sequential MS1 experiments: reference generation and alignment runs. MS1 spectra from a reference LC–MS run are binned and compressed into a spectral matrix. During subsequent runs, each incoming MS1 scan is processed identically and cross‐correlated with reference scans over a defined time window (±10 min). The time difference at the maximum correlation yields the retention shift. Full‐width at half‐maximum on the correlation peak defines left and right uncertainty bounds, which adjust the start and stop times in the inclusion list.

• Mass spectrometer: modified Orbitrap Exploris 480
• MS settings: mass range 350–1200 m/z, resolution 15 k for reference, 60 k for RT alignment, AGC targets 1e6 and 2e6, max IT auto
• Chromatography: Thermo Scientific EASY-Spray ES906 column (2 μm, 150 mm × 150 μm), Vanquish Neo UHPLC, direct injection, 1 μL sample, 1 μL/min flow, 30 min gradients
• Standards: Pierce HeLa digest and PRTC peptides spiked at 50 fmol/μL

Main Results and Discussion

Real‐time alignment of PRTC standards under gradient shifts showed excellent agreement between estimated and actual retention time corrections, with slope and apex coverage consistently matching across conditions. Application to ~2500 HeLa digest peptides achieved over 95% coverage within recalculated windows for all tested gradients and starting windows (6–30 s). Window broadening due to uncertainty bounds remained within 6–9 s. Processing times were compatible with real‐time acquisition, maintaining targeted MS2 scheduling without significant overhead.

Benefits and Practical Applications

• Eliminates the need for frequent spiking of calibration standards
• Maintains narrow retention time windows, increasing the number of measurable targets
• Enhances reproducibility and quantification accuracy in large proteomics cohorts
• Adapts dynamically to column aging and gradient fluctuations

Future Trends and Possibilities

• Scaling to larger cohorts and diverse sample types to further validate robustness
• Integration with other mass spectrometer platforms and acquisition modes
• Algorithmic refinements to reduce uncertainty bounds and processing time
• Combination with isotope‐labeled standards and multiplexed workflows for QA/QC

Conclusion

The implementation of MS1‐based real‐time retention time alignment on an Orbitrap Exploris 480 enables highly accurate, on‐the‐fly correction of chromatographic drift without spiking external standards. The method achieves over 97% retention time coverage under diverse gradient conditions, supporting narrow scheduling windows and improved quantitative performance in targeted proteomics.

References

1. Remes PM, Yip P, MacCoss MJ. Highly Multiplex Targeted Proteomics Enabled by Real-Time Chromatographic Alignment. Analytical Chemistry. 2020;92(17):11809–11817. DOI:10.1021/acs.analchem.0c02075