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Scaling-up low input spatial proteomics using Evosep Whisper Zoom on the timsTOF Ultra 2

Abstract #305 Posted on28 March 20259 May 2025 9 May 2025 20h29

Abstract

Throughput and scalability are important when it comes to spatially resolved analyses of even smaller tissue pieces using proteomics to understand cellular heterogeneity in a disease context. Here, we demonstrate applicability of the Evosep Whisper Zoom methods for speeding up label-free deep visual proteomics on FFPE preserved tissue analysis of up to 120 samples per day (SPD) with data acquisition on the timsTOF Ultra 2.
FFPE preserved tissue contours from 5 µm thick mouse liver and human tonsil were cut by laser capture microdissection and transferred into 384 well plate or proteoCHIP EVO-96. Samples were transferred by centrifugation (proteoCHIP EVO-96) or transferred manually onto Evotips, separated in Whisper Zoom 120, 80, 40 and 20SPD, analyzed on a timsTOF Ultra 2 in dia-PASEF and processed with Spectronaut 19.
Mouse liver tissue contours analyzed in Whisper Zoom 120, 80, 40 and 20SPD demonstrated that protein group IDs more dependent on column length rather than gradient length with 120 and 80SPD yielding up to 3,000 protein groups and 40 and 20SPD yielded about 4,000 protein IDs.
Contours from T-cell rich niches, B-cell rich niches, mixed B- and T-cell zones as well as epithelial cell zones from FFPE preserved human tonsil analyzed in 120SPD yielded proteome depths of about 3,500 protein groups, demonstrating clear differences between the 4 selected zones. Typical marker proteins for T-cells (CD3), B-cells (CD19) and epithelial cells (CDH1) where identified.

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Throughput and scalability are important when it comes to spatially resolved analyses of even smaller tissue pieces using proteomics to understand cellular heterogeneity in a disease context. Here, we demonstrate applicability of the Evosep Whisper Zoom methods for speeding up label-free deep visual proteomics on FFPE preserved tissue analysis of up to 120 samples per day (SPD) with data acquisition on the timsTOF Ultra 2.
FFPE preserved tissue contours from 5 µm thick mouse liver and human tonsil were cut by laser capture microdissection and transferred into 384 well plate or proteoCHIP EVO-96. Samples were transferred by centrifugation (proteoCHIP EVO-96) or transferred manually onto Evotips, separated in Whisper Zoom 120, 80, 40 and 20SPD, analyzed on a timsTOF Ultra 2 in dia-PASEF and processed with Spectronaut 19.
Mouse liver tissue contours analyzed in Whisper Zoom 120, 80, 40 and 20SPD demonstrated that protein group IDs more dependent on column length rather than gradient length with 120 and 80SPD yielding up to 3,000 protein groups and 40 and 20SPD yielded about 4,000 protein IDs. 
Contours from T-cell rich niches, B-cell rich niches, mixed B- and T-cell zones as well as epithelial cell zones from FFPE preserved human tonsil analyzed in 120SPD yielded proteome depths of about 3,500 protein groups, demonstrating clear differences between the 4 selected zones. Typical marker proteins for T-cells (CD3), B-cells (CD19) and epithelial cells (CDH1) where identified.

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