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DOE OSTI · 2564881

Robust collection and processing for label-free single voxel proteomics

Kitata, Reta Birhanu [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000289737679)·Velickovic, Marija [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000336645719)·Xu, Zhangyang [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000203149244)·Zhao, Rui [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)]·Scholten, David [Northwestern University, Chicago, IL (United States)]·Chu, Rosalie K. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000174287647)·Orton, Daniel J. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000202536859)·Chrisler, William B. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)]·Zhang, Tong [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)]·Mathews, Jeremy V. [Northwestern University, Chicago, IL (United States)]·Bumgarner, Benjamin M. [University of Florida, Gainesville, FL (United States)]·Gursel, Demirkan B. [Northwestern University, Chicago, IL (United States)]·Moore, Ronald J. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000328062855)·Piehowski, Paul D. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000151082227)·Liu, Tao [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000195296550)·Smith, Richard D. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000223812349)·Liu, Huiping [Northwestern University, Chicago, IL (United States)] (ORCID:0000000348227995)·Wasserfall, Clive H. [University of Florida, Gainesville, FL (United States)] (ORCID:0000000235228932)·Tsai, Chia-Feng [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000265146911)·Shi, Tujin [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000255923588)
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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 $\underline{w}et$ $\underline{c}ollection$ of single microscale tissue voxels and $\underline{S}urfactant$$-assisted$ $\underline{O}ne$-$\underline{P}ot$ voxel processing method termed wcSOP for robust label-free single voxel proteomics. wcSOP capitalizes on buffer droplet-assisted wet collection of a single voxel dissected by LCM into the PCR tube cap and MS-compatible surfactant-assisted one-pot voxel processing in the collection cap. This convenient method allows reproducible label-free quantification of ~900 and ~4,600 proteins for single voxels at 20 µm × 20 µm × 10 µm (close to single cells) and 200 µm × 200 µm × 10 µm (~100 cells) from fresh frozen human spleen tissue, respectively. 100s-1000s of protein signatures were spatially resolved between spleen red and white pulp regions depending on the voxel size. Region-specific signaling pathways were enriched from single voxel proteomics data. To evaluate its broad applicability, we applied wcSOP-MS to two commonly accessible, OCT-embedded and FFPE, human archived tissues. It enabled to identify spatially resolved proteome changes and enriched pathways between diseased (breast cancer tumor or AD amyloid plaque) and adjacent normal regions. Antibody-based CODEX and IHC imaging validated label-free MS quantitation for single voxel analysis. The wcSOP-MS method paves the way for routine robust single voxel proteomics and spatial proteomics.

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59 BASIC BIOLOGICAL SCIENCESLCMSpatial proteomicsregion of interestsingle voxel proteomicswcSOP-MS

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Kitata, Reta Birhanu [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000289737679), Velickovic, Marija [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000336645719), Xu, Zhangyang [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000203149244), Zhao, Rui [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)], Scholten, David [Northwestern University, Chicago, IL (United States)], Chu, Rosalie K. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000174287647), Orton, Daniel J. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000202536859), Chrisler, William B. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)], Zhang, Tong [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)], Mathews, Jeremy V. [Northwestern University, Chicago, IL (United States)], Bumgarner, Benjamin M. [University of Florida, Gainesville, FL (United States)], Gursel, Demirkan B. [Northwestern University, Chicago, IL (United States)], Moore, Ronald J. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000328062855), Piehowski, Paul D. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000151082227), Liu, Tao [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000195296550), Smith, Richard D. [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000223812349), Liu, Huiping [Northwestern University, Chicago, IL (United States)] (ORCID:0000000348227995), Wasserfall, Clive H. [University of Florida, Gainesville, FL (United States)] (ORCID:0000000235228932), Tsai, Chia-Feng [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000265146911), Shi, Tujin [Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)] (ORCID:0000000255923588). 2025-01-13. Robust collection and processing for label-free single voxel proteomics. https://doi.org/10.1038/s41467-024-54643-x

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