Coupling Microdroplet-Based Sample Preparation, Multiplexed Isobaric Labeling, and Nanoflow Peptide Fractionation for Deep Proteome Profiling of the Tissue Microenvironment

Veličković, Marija; Fillmore, Thomas L.; Attah, Isaac Kwame; Posso, Camilo; Pino, James C.; Zhao, Rui; Williams, Sarah M.; Veličković, Dušan; Jacobs, Jon M.; Burnum-Johnson, Kristin E.; Zhu, Ying; Piehowski, Paul D.

doi:10.1021/acs.analchem.4c00523

Coupling Microdroplet-Based Sample Preparation, Multiplexed Isobaric Labeling, and Nanoflow Peptide Fractionation for Deep Proteome Profiling of the Tissue Microenvironment

Journal Article · Thu Aug 01 00:00:00 EDT 2024 · Analytical Chemistry

DOI:https://doi.org/10.1021/acs.analchem.4c00523· OSTI ID:2482231

Veličković, Marija ^[1]; Fillmore, Thomas L. ^[1]; ^[2]; Posso, Camilo ^[2]; Pino, James C. ^[2]; Zhao, Rui ^[1]; Williams, Sarah M. ^[1]; ^[1]; Jacobs, Jon M. ^[1]; ^[1]; ^[1]; ^[1]

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

There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity in a cell-type-specific manner to better understand and predict the function of complex biological systems, such as human organs. Existing spatially-resolved proteomics technologies cannot provide deep proteome coverage due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (Microdroplet Processing in One pot for Trace Samples), multiplexed isobaric labelling, and a nanoflow peptide fractionation approach. The integrated workflow allowed us to maximize proteome coverage of laser-isolated tissue samples containing nanogram level of proteins. We demonstrated that the deep spatial proteomics platform can quantify more than 5,000 unique proteins from a small-sized human pancreatic tissue pixel (~60,000 µm²) and differentiate unique protein abundance patterns in pancreas. Further, the use of microPOTS chip eliminated the requirement for advanced microfabrication capabilities and specialized nanoliter liquid handling equipment, making it more accessible to proteomic laboratories.

View Accepted Manuscript (DOE)

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

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 2482231

Alternate ID(s):: OSTI ID: 2471609

Report Number(s):: PNNL-SA--194410

Journal Information:: Analytical Chemistry, Journal Name: Analytical Chemistry Journal Issue: 32 Vol. 96; ISSN 0003-2700

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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