Examining Nanoscale LC Reproducibility with Coupling of the ACQUITY UPLC M- Class System to SELECT SERIES Cyclic IMS

Significance of the Topic

High-performance nanoscale liquid chromatography coupled with advanced mass spectrometry is critical for proteomics, especially when analyzing limited or precious biological samples. Ensuring reproducible retention times, signal intensities and mass accuracy underpins reliable protein identification and quantitation across large sample cohorts. Robustness in these metrics supports downstream statistical analyses and biomarker discovery in clinical and research settings.

Objectives and Study Overview

This application note evaluates the reproducibility of a nanoscale LC–ion mobility MS platform over an extended 23-day experiment. A standard E. coli tryptic digest served as a quality control (QC) sample, injected at regular intervals among ovarian cancer cell line digests. The goal was to quantify retention time, signal intensity, mass accuracy and protein identification consistency when using an ACQUITY UPLC M-Class System coupled to a SELECT SERIES™ Cyclic IMS operating in data-independent acquisition mode.

Methodology and Used Instrumentation

Sample Preparation:

• Waters MPDS E. coli tryptic digest diluted for 100 ng injections.

Liquid Chromatography:

• System: ACQUITY UPLC M-Class
• Trapping column: Symmetry C18, 180 µm×20 mm, 5 µm
• Analytical column: HSS T3, 75 µm×250 mm, 1.8 µm
• Flow: 300 nL/min; 5 – 35 % acetonitrile over 90 min; column at 40 °C.

Mass Spectrometry:

• System: SELECT SERIES Cyclic IMS Q-ToF
• Ionization: ESI positive
• Mode: Ion mobility-enabled HDMSE
• Mass range: 50–2000 amu; resolution 50,000 FWHM; IMS resolution ~65 FWHM
• Acquisition: 0.5 s integration; reference lockspray every 120 s

Data Processing:

• Software: MassLynx; ProteinLynx Global Server; Tibco Spotfire
• Database: Reviewed UniProt E. coli
• FDR threshold: 4 %

Main Results and Discussion

Retention Time Reproducibility:

• Five peptides across a 95 min gradient displayed CVs of ~1 %.

Signal Intensity Reproducibility:

• Twelve peptides across intensity range showed CVs under 20 %.

Mass Accuracy:

• 82 % of 275k peptide measurements were within ±2 ppm of theoretical mass.

Protein and Peptide Identifications:

• Average ~1,100 proteins and ~22,000 peptides per QC injection.
• Over 1,000 proteins consistently identified in ≥75 % of runs.

Sequence Coverage:

• Twelve proteins showed 55 – 93 % coverage with CVs between 3 – 5 %.

Dynamic Range:

• Consistent identification spanning four orders of magnitude in fragment-ion signal.

Benefits and Practical Applications

• Reliable retention time alignment supports large-cohort studies.
• Consistent intensity and identification metrics enable quantitative comparisons and biomarker validation.
• High mass accuracy and ion mobility resolution improve identification confidence.

Future Trends and Applications

Integration of cyclic ion mobility with data-independent workflows is anticipated to further enhance peak capacity and selectivity. Ongoing developments in column chemistries and microflow interfaces may boost throughput while retaining nanoscale sensitivity. Applications will extend into single-cell proteomics, post-translational modification mapping and systems biology studies where reproducibility and depth are paramount.

Conclusion

The combined ACQUITY UPLC M-Class nanoscale LC system and SELECT SERIES Cyclic IMS mass spectrometer delivered exceptional reproducibility in retention time, signal intensity and mass accuracy over 23 days. These robust performance metrics translated into consistent protein identification rates and broad dynamic range, reinforcing the statistical validity of large-scale proteomic experiments.

References

1. Chris Hughes, Lee A. Gethings, Robert S. Plumb. Qualitative and Quantitative Performance of Cyclic IMS in Nanoscale Proteomic Experiments. Waters Application Note 720007381, 2021.