Enhancing Sensitivity in Targeted Single-Cell Proteomics by Coupling a Dual Ion Funnel Interface with Triple Quadrupole Mass Spectrometer

Single-cell proteomics (SCP) has emerged as a powerful
approach
for understanding cellular heterogeneity and biological processes
at unprecedented resolution. However, the extremely limited protein
content of individual cells (femtogram to picogram levels) pushes
current mass spectrometry instrumentation to its sensitivity limits,
creating a critical analytical bottleneck. While selected reaction
monitoring (SRM) using triple quadrupole (QqQ) instruments offers
advantages in sensitivity and reproducibility for targeted proteomics
quantification, SRM still struggles with sensitivity for quantification
of moderate- or low-abundance proteins from single-cell sample amounts.
Here, we report the development and systematic evaluation of a dual
ion funnel interface designed to address the sensitivity limitation
by significantly enhancing ion transmission efficiency in commercial
QqQ mass spectrometers. The dual ion funnel interface, composed of
a curved S-funnel followed by a conventional ion funnel, improves
ion transmission efficiency while reducing chemical noise through
selective ion focusing. The performance of the dual ion funnel interface
was systematically compared to standard interface on a TSQ Vantage
platform across samples with different levels of complexity. The dual
funnel interface demonstrated to provide up to 25-fold improvement
in sensitivity across a wide range of protein concentrations in different
biological matrices (low complex mouse macrophage and high complex
human cells). Critically, enhanced sensitivity was accompanied by
increased analytical reproducibility with lower coefficient of variations.
Most importantly, the dual funnel interface enabled reliable quantification
of low-abundance proteins that were barely detectable or not detected
by the standard interface, extending analysis to single-cell equivalent
amounts while maintaining excellent reproducibility. These results
demonstrate that the dual funnel interface addresses the critical
bottleneck in quantitative targeted proteomics, providing a technological
foundation for ultrasensitive targeted SCP that requires both high
sensitivity and robust quantitative performance.