An Ultrasensitive Spatial Tissue Proteomics Workflow Exceeding 100 Proteomes Per Day

Area of Science:

• Proteomics
• Mass Spectrometry
• Tissue Analysis

Background:

• High-resolution spatial tissue proteomes require optimized sample processing, chromatography, and mass spectrometry (MS) acquisition.
• Balancing these factors is crucial for comprehensive proteome coverage.

Purpose of the Study:

• To present an advanced cellenONE protocol for loss-reduced tissue processing.
• To compare Evosep ONE Whisper Zoom gradients and common DIA acquisition schemes on a timsUltra AIP mass spectrometer.
• To evaluate the impact of tissue type, gradient length, and sample amount on proteome coverage.

Main Methods:

• Utilized an advanced cellenONE protocol for tissue processing.
• Employed Evosep ONE Whisper Zoom gradients (20, 40, 80, 120 samples per day).
• Used three common data-independent acquisition (DIA) schemes on a timsUltra AIP mass spectrometer.

Main Results:

• Tissue type, gradient length, and sample amount significantly influence proteome coverage.
• Increased tissue sampling yielded diminishing returns for short gradients (120/80 SPD) but substantial gains for longer gradients (40/20 SPD).
• Quantified over 7,500 proteins from 0.5 nL of tonsil T-cell niches using longer gradients and higher tissue amounts; 3,200 proteins were quantified from 0.04 nL with the 120 SPD method.

Conclusions:

• The developed workflow enables the preparation and acquisition of over 100 high-quality spatial tissue proteomes daily.
• This facilitates cohort-size spatial tissue proteomics for translational research.
• Demonstrated application to oral cavity squamous cell carcinoma, revealing disease pathways and region-specific changes.