Spatially Resolved Top-Down Proteomics of Tissue Sections Based on a Microfluidic Nanodroplet Sample Preparation Platform

Liao, Yen-Chen; Fulcher, James M.; Degnan, David J.; Williams, Sarah M.; Bramer, Lisa M.; Veličković, Dušan; Zemaitis, Kevin J.; Veličković, Marija; Sontag, Ryan L.; Moore, Ronald J.; Paša-Tolić, Ljiljana; Zhu, Ying; Zhou, Mowei

doi:10.1016/j.mcpro.2022.100491

Spatially Resolved Top-Down Proteomics of Tissue Sections Based on a Microfluidic Nanodroplet Sample Preparation Platform

Journal Article · Mon Jan 02 00:00:00 EST 2023 · Molecular and Cellular Proteomics

DOI:https://doi.org/10.1016/j.mcpro.2022.100491· OSTI ID:1964103

^[1]; ^[1]; ^[2]; ^[1]; ^[2]; Veličković, Dušan ^[1]; ^[1]; Veličković, Marija ^[1]; Sontag, Ryan L. ^[2]; Moore, Ronald J. ^[2]; Paša-Tolić, Ljiljana ^[1]; Zhu, Ying ^[1]; ^[1]

Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Conventional proteomics measures the averaged signal from mixed cell populations or bulk tissues, leading to the dilution of significant changes in subpopulations of cells that might serve as important biomarkers. Recent developments in bottom-up proteomics have enabled spatial mapping of cellular heterogeneity in tissue microenvironments. However, bottom-up proteomics cannot precisely infer the abundance changes of intact proteins, which are presented as proteoforms. Herein, we described a spatially resolved top-down proteomics (TDP) platform for proteoform identification and quantification directly on thin tissue sections. The spatial TDP platform consisted of a nanoPOTS (nanodroplet Processing in One pot for Trace Samples)-based sample preparation system and an LCM (laser capture microdissection)-based cell isolation system. We improved the nanoPOTS sample preparation by adding benzonase in the extraction buffer to enhance the coverage of nucleus proteins. Using ~200 cultured cells as model samples, the improved approach increased proteoform identifications from 493 to 700; newly identified proteoforms primarily corresponded to nuclear proteins. To demonstrate the spatial TDP platform in tissue samples, we analyzed LCM-isolated tissue voxels from rat brain cortex and hypothalamus regions. We quantified 426 proteoforms by combining identifications from TopPIC and TDPortal with the quantitation from ProMex. Several proteoforms corresponding to the same gene exhibited mixed abundance profiles between two tissue regions, suggesting potential PTM-specific spatial distributions. The spatial TDP workflow has prospects for biomarker discovery at proteoform level from small tissue sections.

View Accepted Manuscript (DOE)

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: National Institutes of Health (NIH); National Resource for Translational and Developmental Proteomics; Sherman Fairchild Foundation; USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 1964103

Report Number(s):: PNNL-SA-174522

Journal Information:: Molecular and Cellular Proteomics, Journal Name: Molecular and Cellular Proteomics Journal Issue: 2 Vol. 22; ISSN 1535-9476

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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Spatially Resolved Proteome Mapping of Laser Capture Microdissected Tissue with Automated Sample Transfer to Nanodroplets

Journal Article · Sun Jun 24 00:00:00 EDT 2018 · Molecular and Cellular Proteomics · OSTI ID:1503624

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59 BASIC BIOLOGICAL SCIENCES
laser capture microdissection
nanoPOTS
proteoform
quantitation
spatial proteomics
top-down proteomics

Spatially Resolved Top-Down Proteomics of Tissue Sections Based on a Microfluidic Nanodroplet Sample Preparation Platform

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