Qualitative and Quantitative Performance of Cyclic IMS in Nanoscale Proteomic Experiments

Importance of the Topic

Analytical advances in proteomics require instruments with high sensitivity, mass and ion mobility resolution to characterize complex biological samples. The ability to separate ions by mobility adds an orthogonal dimension to traditional LC-MS workflows, enhancing the depth of proteome coverage and quantitative accuracy at nanoscale sample loads.

Objectives and Overview

This study evaluates the qualitative and quantitative performance of the SELECT SERIES Cyclic IMS coupled to a nanoscale ACQUITY UPLC M-Class system. Using single-pass ion mobility separations in HDMSE acquisition, the work compares protein and peptide identification rates, dynamic range, mass accuracy, and reproducibility against prior QTof iterations.

Methodology

• Sample preparation: Tryptic digests of E coli and human K562 cell lysates loaded at 10–100 ng on column; isotopologue peptide mixture spiked into E coli background for quantitation.
• Chromatography conditions: Nano flow 300 nL/min, Symmetry trap and HSS T3 analytical column at 35 °C, gradient 5–35% acetonitrile over 90–240 minutes.
• Acquisition parameters: HDMSE data-independent acquisition, mass range 50–2000 Da, integration time 0.5 s, lock mass Glu-Fibrinopeptide B every 120 s.
• Data processing: MassLynx, ProteinLynx Global Server, Progenesis QI, Uniprot reviewed databases, FDR thresholds at 1% and 4%.

Used Instrumentation

• ACQUITY UPLC M-Class nano LC system
• SELECT SERIES Cyclic IMS QTof mass spectrometer
• MassLynx MS acquisition software
• ProteinLynx Global Server and Progenesis QI for data analysis

Main Results and Discussion

• Identification depth: Optimal loading of 50–75 ng yielded ~4500 proteins at 1% FDR with a 240-minute gradient; E coli reached ~1250 proteins at 50 ng.
• Mass accuracy: Over 90% of peptides measured within ±5 ppm.
• Dynamic range: Spanned five orders of magnitude for identification intensities and quantitative series.
• Reproducibility: Technical replicates achieved r2 ≈ 0.999 for protein intensities; 3602 proteins consistently identified in two out of three runs at 1% FDR.
• Quantitation: Stable isotope-labeled peptide series demonstrated linear response from 10 amol to 100 fmol on column.

Benefits and Practical Applications

• Enhanced sensitivity and selectivity for profiling limited or low-abundance samples.
• Improved mass and ion mobility resolution increases confident identifications in complex matrices.
• Extended dynamic and quantitative range supports robust biomarker discovery and targeted assays.
• High reproducibility across extended runs facilitates large-scale cohort studies.

Future Trends and Opportunities

• Multi-pass ion mobility separations to boost resolution and peak capacity further.
• Integration with advanced data-independent acquisition strategies for deeper proteome coverage.
• Application in single-cell and sub-nanogram proteomics leveraging nanoscale LC flows.
• AI-driven data analysis to exploit high-dimensional mobility and mass spectral information.

Conclusion

The SELECT SERIES Cyclic IMS coupled with nanoscale UPLC delivers significant improvements in proteomic analyses, offering higher identification rates, exceptional mass accuracy, broad dynamic range, and outstanding run-to-run reproducibility. These capabilities advance both qualitative discovery and quantitative workflows, paving the way for more sensitive and robust proteomic investigations.

Reference

1. C J Hughes, L A Gethings. Characteristics of Proteomics Experiments Performed on the SYNAPT XS QTof Mass Spectrometer. Waters Application Note 720006670EN, 2019.