EASY-Spray and double nanoViper PepMap Neo columns - PRODUCT SPECIFICATIONS

Importance of the Topic

Peptide mapping via low-flow chromatography is essential for obtaining high-quality data in bottom-up proteomics workflows. However, conventional setups can be time-consuming and prone to variability. The combination of EASY-Spray and double nanoViper PepMap Neo columns simplifies system connectivity, enhances reproducibility, and improves chromatographic separation, thereby boosting proteome coverage.

Objectives and Overview

This study evaluates the performance and robustness of Thermo Scientific PepMap Neo columns in conjunction with the Vanquish Neo UHPLC System and Orbitrap Exploris 480 mass spectrometer. Key goals include assessing peptide and protein identification consistency across multiple columns, and monitoring chromatographic stability over hundreds of injections.

Methodology

A direct injection workflow was employed using HeLa cell digests. Four EASY-Spray PepMap Neo columns (75 µm I.D. × 750 mm) were tested for inter-column reproducibility of peptide and protein identification and quantification. A ruggedness study involved 500 consecutive injections on a 75 µm I.D. × 500 mm double nanoViper PepMap Neo column, tracking retention time, peak width, peak symmetry, and system pressure.

Used Instrumentation

• Thermo Scientific Vanquish Neo UHPLC System
• Thermo Scientific Orbitrap Exploris 480 Mass Spectrometer
• EASY-Spray PepMap Neo columns, 2 µm C18, 100 Å, 75 µm × 750 mm with integrated glass emitter
• Double nanoViper PepMap Neo columns, 2 µm C18, 100 Å, 75 µm × 500 mm

Main Results and Discussion

Across four EASY-Spray columns, peptide identifications ranged from ~77 000 to ~94 000 and protein identifications remained around 7 000, with quantification showing similar consistency. Over 500 injections on a double nanoViper column, retention times exhibited minimal drift, peak widths and asymmetry remained stable, and system pressure stayed within expected ranges. These results highlight the efficacy of high-pressure packing and zero-dead-volume connections in delivering reproducible chromatographic performance.

Benefits and Practical Applications

The streamlined connectivity and robust performance of PepMap Neo columns facilitate high-throughput proteomics, support large-scale or multi-site studies, and reduce downtime associated with column handling. Improved peak capacity enables deeper proteome coverage, benefiting applications in pharmaceutical development, biomarker discovery, and quality control.

Future Trends and Potential Applications

Advances may include longer columns, smaller particle sizes, and elevated temperatures to further reduce backpressure and enhance separation. Integration with automated sample preparation and artificial intelligence–driven data analysis could accelerate workflows in single-cell proteomics and post-translational modification studies.

Conclusion

Thermo Scientific EASY-Spray and double nanoViper PepMap Neo columns, combined with the Vanquish Neo UHPLC System and Orbitrap Exploris 480, offer a robust solution for reproducible, high-efficiency peptide mapping. The consistent performance across columns and injections simplifies proteomics workflows and enables comprehensive protein characterization.

References

• Thermo Scientific Technical Note TN74152