The Agilent Nanodapter for Discovery Proteomics Using Nanoflow LC/MS

Importance of the Topic

Nanoflow liquid chromatography–mass spectrometry (LC/MS) is critical in discovery proteomics when sample amounts are low and maximum sensitivity is required. It complements standard flow proteomics by delivering enhanced analyte ionization efficiency and lower detection limits, enabling deep proteome coverage in limited or precious biological samples.

Objectives and Overview of the Study

This technical overview describes the Agilent Infinity UHPLC Nanodapter, a modular add-on that converts an Agilent 1290 Infinity II UHPLC system from standard flow to nanoflow operation. The study evaluates its flexibility, ease of configuration, and analytical performance in discovery proteomics when interfaced with the Agilent G1992A nanoESI source and 6550 iFunnel Q-TOF mass spectrometer.

Methodology and Instrumentation

The Nanodapter adapts the 1290 Infinity II binary pump to deliver 100–1,000 nL/min through 75–150 µm internal diameter columns at pressures up to 1,000 bar. An in-line electronic flow sensor continuously monitors nanoflow rates. Users can switch between two operating modes in under 30 minutes:

• Direct injection mode: optimal for hydrophilic peptides via a guard column and analytical column (75 µm × 2 cm and 75 µm × 25 cm C18, respectively).
• Trapping mode: faster loading of larger volumes using a trap column before analytical separation.

Configuration was tested on a human K562 cell extract digest (1 µg) using a 120-minute gradient. Key instrument parameters included:

• LC gradient: 3→70 % B over 120 min at ~320 nL/min on column.
• Mass spectrometry: Agilent 6550 iFunnel Q-TOF in Extended Dynamic Range mode (m/z 300–1,700), 8 spectra/s MS and 3 spectra/s MS/MS acquisition, top-20 data-dependent selection, 1.3 amu isolation width, dynamic exclusion enabled.

Main Results and Discussion

Triplicate analyses demonstrated high chromatographic reproducibility, with a median peak width at half maximum of 13 seconds and consistent base peak chromatograms. Across three runs, a cumulative total of 20,735 unique peptide sequences and 2,833 proteins were identified, with each run yielding on average 16,025 peptides and 2,437 proteins. These metrics confirm the Nanodapter’s ability to provide both sensitivity and robustness in low-abundance proteome profiling.

Benefits and Practical Applications

The Infinity UHPLC Nanodapter offers several advantages:

• Seamless switching between standard and nanoflow without additional pumps.
• Enhanced sensitivity for limited-sample discovery workflows.
• Broad compatibility with commercially available nanoflow columns.
• Continuous flow monitoring for improved run reliability.

This configuration is particularly useful for biomarker discovery, post-translational modification mapping, and any application requiring deep proteome interrogation of scarce samples.

Future Trends and Applications

Looking ahead, combining nanoflow LC/MS with real-time data processing and advanced ion mobility separation could further boost proteome coverage and throughput. Integration with automated sample preparation and AI-driven method optimization may streamline workflows in clinical proteomics, pharmaceutical research, and systems biology.

Conclusion

The Agilent Infinity UHPLC Nanodapter transforms a standard UHPLC system into a flexible nanoflow platform, marrying sensitivity with robustness. Its easy configuration and proven proteomic performance make it a valuable tool for laboratories aiming to maximize discovery potential from limited sample amounts.

Reference

1. Jet Stream Proteomics for Sensitive and Robust Standard Flow LC/MS, Agilent Technologies, publication number 5991-5687EN.
2. Maximize Nanoflow LC/MS Performance Using the Flexible, Easy-to-configure Agilent nanoESI Source, Agilent Technologies, publication number 5991-1041EN.