# An Ultrasensitive Spatial Tissue Proteomics Workflow Exceeding 100 Proteomes Per Day

## TL;DR

A new workflow enables processing over 100 high-quality spatial tissue proteomes per day, using minimal tissue and revealing disease-specific protein patterns.

## Contribution

An ultrasensitive workflow for high-throughput spatial tissue proteomics, benchmarking Evosep gradients and DIA-PASEF settings.

## Key findings

- Over 100 high-quality spatial tissue proteomes can be generated daily using the optimized workflow.
- Up to 7500 proteins can be quantified from as little as 0.04 nL of formalin-fixed paraffin-embedded tissue.
- The workflow revealed regional heterogeneity in oral squamous cell carcinoma proteomes.

## Abstract

Achieving high-resolution spatial tissue proteomes requires careful balancing and integration of optimized sample processing, chromatography, and MS acquisition. Here, we present an advanced cellenONE protocol for loss-reduced tissue processing and compare all Evosep ONE Whisper Zoom gradients (20, 40, 80, and 120 samples per day), along with three common data-independent acquisition schemes on a timsUltra athena ion processor mass spectrometer. We found that tissue type was as important as gradient length and sample amount in determining proteome coverage. Moreover, the benefit of increased tissue sampling was gradient- and dynamic range-dependent. Analyzing mouse liver, a high dynamic range tissue, over tenfold more tissue sampling led to only ∼30% gain in protein identification for short gradients (120 samples per day (SPD) and 80 SPD). However, even the lowest tested tissue amount (0.04 nl) yielded 3200 reproducibly quantified proteins for the 120 SPD method. Longer gradients (40 SPD and 20 SPD) instead significantly benefited from more tissue sampling, quantifying over 7500 proteins from 0.5 nl of tonsil T-cell niches. Finally, we applied our workflow to a rare squamous cell carcinoma of the oral cavity, uncovering disease-associated pathways and region-specific protein level changes. Our study demonstrates that more than 100 high-quality spatial tissue proteomes can be prepared and acquired daily, laying a strong foundation for cohort-size spatial tissue proteomics in translational research.

•Optimized workflow enables >100 spatial tissue proteomes per day.•Deep proteomic coverage - up to 7500 proteins - from just 0.04 to 0.5 nL FFPE tissue.•Benchmarking Evosep Whisper Zoom gradients and optimized DIA-PASEF settings.•Spatial proteomics of oral squamous cell carcinoma reveals regional heterogeneity.

Optimized workflow enables >100 spatial tissue proteomes per day.

Deep proteomic coverage - up to 7500 proteins - from just 0.04 to 0.5 nL FFPE tissue.

Benchmarking Evosep Whisper Zoom gradients and optimized DIA-PASEF settings.

Spatial proteomics of oral squamous cell carcinoma reveals regional heterogeneity.

This study presents an ultrasensitive spatial tissue proteomics workflow capable of generating over 100 high-quality proteomes per day. By optimizing cellenONE-based sample processing and evaluating Evosep ONE Whisper Zoom gradients with a timsUltra AIP mass spectrometer, the workflow achieves high quality proteomes from as little as 0.04 nL of tissue. Applied to liver, tonsil, and oral squamous cell carcinoma samples, it revealed tissue-specific proteome patterns and intratumoral heterogeneity for advanced throughput spatial tissue proteomics.