Robust collection and processing for label-free single voxel proteomics

Reta Birhanu Kitata, Marija Velickovic, Zhangyang Xu, Rui Zhao, David Scholten, Rosalie K. Chu, Daniel J. Orton, William B. Chrisler, Tong Zhang, Jeremy V. Mathews, Benjamin M. Bumgarner, Demirkan B. Gursel, Ronald J. Moore, Paul D. Piehowski, Tao Liu, Richard D. Smith, Huiping Liu, Clive H. Wasserfall, Chia-Feng Tsai and Tujin Shi ()
Additional contact information
Reta Birhanu Kitata: Pacific Northwest National Laboratory
Marija Velickovic: Pacific Northwest National Laboratory
Zhangyang Xu: Pacific Northwest National Laboratory
Rui Zhao: Pacific Northwest National Laboratory
David Scholten: Feinberg School of Medicine, Northwestern University
Rosalie K. Chu: Pacific Northwest National Laboratory
Daniel J. Orton: Pacific Northwest National Laboratory
William B. Chrisler: Pacific Northwest National Laboratory
Tong Zhang: Pacific Northwest National Laboratory
Jeremy V. Mathews: Feinberg School of Medicine, Northwestern University
Benjamin M. Bumgarner: College of Medicine, University of Florida
Demirkan B. Gursel: Feinberg School of Medicine, Northwestern University
Ronald J. Moore: Pacific Northwest National Laboratory
Paul D. Piehowski: Pacific Northwest National Laboratory
Tao Liu: Pacific Northwest National Laboratory
Richard D. Smith: Pacific Northwest National Laboratory
Huiping Liu: Feinberg School of Medicine, Northwestern University
Clive H. Wasserfall: College of Medicine, University of Florida
Chia-Feng Tsai: Pacific Northwest National Laboratory
Tujin Shi: Pacific Northwest National Laboratory

Nature Communications, 2025, vol. 16, issue 1, 1-15

Abstract: Abstract With advanced mass spectrometry (MS)-based proteomics, genome-scale proteome coverage can be achieved from bulk tissues. However, such bulk measurement lacks spatial resolution and obscures tissue heterogeneity, precluding proteome mapping of tissue microenvironment. Here we report an integrated wet collection of single microscale tissue voxels and Surfactant-assisted One-Pot voxel processing method termed wcSOP for robust label-free single voxel proteomics. wcSOP capitalizes on buffer droplet-assisted wet collection of single voxels dissected by LCM to the tube cap and SOP voxel processing in the same collection cap. This method enables reproducible, label-free quantification of approximately 900 and 4600 proteins for single voxels at 20 µm × 20 µm × 10 µm (~1 cell region) and 200 µm × 200 µm × 10 µm (~100 cell region) from fresh frozen human spleen tissue, respectively. It can reveal spatially resolved protein signatures and region-specific signaling pathways. Furthermore, wcSOP-MS is demonstrated to be broadly applicable for OCT-embedded and FFPE human archived tissues as well as for small-scale 2D proteome mapping of tissues at high spatial resolutions. wcSOP-MS may pave the way for routine robust single voxel proteomics and spatial proteomics.

Date: 2025
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DOI: 10.1038/s41467-024-54643-x

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