OptiFlow™ Interface for TripleTOF® 6600 System

Importance of the Topic

In modern omics and mass spectrometry core facilities, the ability to alternate rapidly between nano- and microflow liquid chromatography workflows is critical for balancing high sensitivity analyses with high throughput demands. The OptiFlow Interface combined with the nanoLC 425 System offers a streamlined solution to switch flow regimes in minutes, maintaining vacuum integrity and minimizing downtime.

Study Objectives and Overview

This study evaluates the performance and productivity gains enabled by the OptiFlow Interface paired with a TripleTOF 6600 system. Key goals include demonstrating rapid, tool-free transitions between nanoflow and microflow sources and validating the analytical equivalence or superiority of this interface compared to the legacy nanoDCI setup.

Methodology and Instrumentation

Workflow transitions were achieved by replacing the nanoflow curtain plate and heater with a high-flow plate and source (Turbo V or OptiFlow Source in micro mode) on a universal interface plate without breaking the mass spectrometer vacuum. Comparative tests employed:

• PepCalMix standards at 20 fmol and 20 amol loadings, three replicates each.
• SWATH Acquisition Performance Kit on a complex cell lysate digest, measuring over 10 000 peptides.

Instrumentation included a nanoLC 425 System, OptiFlow Interface, OptiFlow and Turbo V sources, nanoSpray Source, and the TripleTOF 6600 mass spectrometer.

Main Results and Discussion

PepCalMix experiments showed peptide area ratios relative to the nanoDCI interface of ≥ 1, with an average ratio of 1.6 and consistent %CV across replicates. SWATH analyses across two OptiFlow Interfaces and the nanoDCI interface yielded similar peptide area distributions and quantified comparable numbers of peptides and proteins at < 1% FDR and < 20% CV. Cumulative CV curves confirmed that ~ 90% of peptide and protein measurements achieved better than 20% reproducibility, underscoring the robustness of the switchover workflow.

Benefits and Practical Applications

The OptiFlow Interface delivers:

• Rapid, tool-free switching between nano- and microflow LC-MS modes without vacuum breaks.
• Enhanced laboratory throughput by enabling two flow regimes on a single LC system.
• Reliable performance for both high-sensitivity discovery and high-throughput quantitation projects.

Future Trends and Opportunities

Advancements may include integration of automated flow regime selection, expansion to additional mass spectrometry platforms, coupling with differential mobility separation, and incorporation of AI-driven method optimization to further streamline complex omics workflows.

Conclusion

The OptiFlow Interface with nanoLC 425 System significantly improves operational flexibility and productivity in LC-MS laboratories. Validation against the nanoDCI interface demonstrates equal or superior analytical performance, supporting both routine high-throughput and sensitive nanoflow applications on a single platform.

Reference

1. Achieving Very High Reproducibility for Quantitative Proteomics with Nanoflow LC-MS – NanoLC 400 Series System, SCIEX Technical Note RUO-MKT-02-5755-A.
2. Configuring the nanoLC 425 System for Both Nanoflow and Microflow LC-MS Workflows, SCIEX Technical Note RUO-MKT-02-7254-A.
3. Easily Switch from Nanoflow to Microflow with OptiFlow Interface and nanoLC 425 System, SCIEX Application Note.
4. SWATH Acquisition Performance Kit, SCIEX Standard.