High Sensitivity Peptide Analysis Using the 6550 Q-TOF with iFunnel Technology

Importance of the Topic

The sensitivity of peptide analysis critically influences protein identification, profiling, and accurate quantitation in proteomics research. High-performance mass spectrometry platforms that can detect low‐abundance peptides enable deeper insight into complex biological samples and reduce sample consumption.

Objectives and Study Overview

This study evaluates the performance enhancement provided by Agilent’s iFunnel technology in the 6550 Q-TOF mass spectrometer for peptide analysis. Key goals include:

• Comparing sensitivity of Agilent 6550 Q-TOF (with iFunnel) to 6540 Q-TOF (without iFunnel).
• Assessing improvement in full scan MS and targeted MS/MS modes using a synthetic HSA‐derived peptide.
• Demonstrating protein identification limits with BSA digest down to 10 amol on-column.
• Evaluating quantitative performance in a complex yeast lysate matrix over a wide dynamic range.

Methodology and Instrumentation

Chromatography and Ionization:

• Reverse‐phase LC using Agilent 1290 Infinity system (2.1 × 50 mm, 400 µL/min).
• Nano-flow LC/MS with HPLC-Chip/MS interface for BSA and yeast experiments.
• Agilent Jet Stream ESI source with thermal gradient ion focusing.

Mass Spectrometry Platforms:

• Agilent 6540 Q-TOF (no ion funnel).
• Agilent 6550 Q-TOF with dual-stage iFunnel.

Analytical Protocols:

• Full scan MS at 2 Hz and targeted MS/MS at 5 Hz acquisition rates.
• Data-dependent mode for protein identification workflows.
• Selected-product‐ion monitoring in complex matrix for quantitation (10 ppm window).

Main Results and Discussion

Full Scan MS:

• Signal intensity increased from ~3 × 10^5 counts to >3 × 10^6 counts for the doubly charged LVNEVTEFAK peptide—an approximate 10-fold boost.

Targeted MS/MS:

• Extracted ion chromatogram of product ion m/z 937.4625 showed a 7-fold sensitivity gain with the iFunnel Q-TOF.

Protein Identification with BSA Digest:

• Successful identification of BSA at 10 amol on-column using nanoLC/MS, yielding 4–8 unique peptides and >10% sequence coverage.
• Progressive increase in unique peptides and coverage up to 10 fmol input.

Quantitative Performance in Yeast Lysate:

• Linear response from 10 amol to 100 fmol on-column (R^2 = 0.9902).
• Limit of detection of 10 amol for target peptide in presence of 150 ng yeast digest.
• Dynamic range spanning four orders of magnitude.

Benefits and Practical Applications

The enhanced sensitivity of the 6550 iFunnel Q-TOF enables:

• Lower sample requirements for proteomics studies, preserving scarce or precious materials.
• Combined qualitative and quantitative workflows on a single platform.
• Transition from nanoLC to high‐throughput UHPLC methods without compromising detection limits.

Future Trends and Opportunities

Emerging developments leveraging iFunnel sensitivity include:

• Integration with data‐independent acquisition (DIA) to expand proteome coverage.
• Application in single‐cell and low‐sample proteomics.
• Adoption of microflow chromatography for robust, high‐throughput analyses.
• Advanced informatics and AI‐driven spectral interpretation to accelerate discovery.

Conclusion

Agilent’s iFunnel technology in the 6550 Q-TOF delivers a marked enhancement in peptide detection across full scan MS, targeted MS/MS, and data-dependent acquisition modes. This advancement broadens the capabilities of proteomics workflows, enabling deeper analysis of low‐abundance peptides and streamlined quantitative assays on a single, versatile platform.

Reference

1. The 6490 Triple Quad LC/MS Enables the Highest Sensitivity for Peptide Quantitation in Plasma. Agilent Publication, 2010, publication number 5990-6513EN.