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

DOE OSTI · 2471609

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.

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Abstract

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 labeling, and a nanoflow peptide fractionation approach. The integrated workflow allowed us to maximize proteome coverage of laser-isolated tissue samples containing nanogram levels of proteins. We demonstrated that the deep spatial proteomics platform can quantify more than 5000 unique proteins from a small-sized human pancreatic tissue pixel (∼60,000 μm2) and differentiate unique protein abundance patterns in pancreas. Furthermore, the use of the microPOTS chip eliminated the requirement for advanced microfabrication capabilities and specialized nanoliter liquid handling equipment, making it more accessible to proteomic laboratories.

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY Chemistry Fractionation Labeling Mass spectrometry Peptides and proteins Proteomics

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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.. 2024-08-01. Coupling Microdroplet-Based Sample Preparation, Multiplexed Isobaric Labeling, and Nanoflow Peptide Fractionation for Deep Proteome Profiling of the Tissue Microenvironment. https://doi.org/10.1021/acs.analchem.4c00523

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