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    Sensitive Top-Down Proteomics Analysis of a Low Number of Mammalian Cells Using a Nanodroplet Sample Processing Platform
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    • Mowei Zhou
      Mowei Zhou
      Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, United States
      More by Mowei Zhou
      Orcidhttp://orcid.org/0000-0003-3575-3224
    • Naomi Uwugiaren
      Naomi Uwugiaren
      International Centre for Cancer Vaccine Science, University of Gdansk, Gdansk, Poland
      More by Naomi Uwugiaren
    • Sarah M. Williams
      Sarah M. Williams
      Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, United States
      More by Sarah M. Williams
    • Ronald J. Moore
      Ronald J. Moore
      Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99354, United States
      More by Ronald J. Moore
      Orcidhttp://orcid.org/0000-0003-2806-2855
    • Rui Zhao
      Rui Zhao
      Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, United States
      More by Rui Zhao
    • David Goodlett
      David Goodlett
      International Centre for Cancer Vaccine Science, University of Gdansk, Gdansk, Poland
      Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, Maryland 21201, United States
      More by David Goodlett
    • Irena Dapic
      Irena Dapic
      International Centre for Cancer Vaccine Science, University of Gdansk, Gdansk, Poland
      More by Irena Dapic
    • Ljiljana Paša-Tolić
      Ljiljana Paša-Tolić
      Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, United States
      More by Ljiljana Paša-Tolić
    • Ying Zhu*
      Ying Zhu
      Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, United States
      *[email protected]
      More by Ying Zhu
      Orcidhttp://orcid.org/0000-0002-5416-0566
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    Analytical Chemistry

    Cite this: Anal. Chem. 2020, 92, 10, 7087–7095
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    https://doi.org/10.1021/acs.analchem.0c00467
    Published May 6, 2020
    Copyright © 2020 American Chemical Society
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    Abstract

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    Top-down proteomics is a powerful tool for characterizing genetic variations and post-translational modifications at intact protein level. However, one significant technical gap of top-down proteomics is the inability to analyze a low amount of biological samples, which limits its access to isolated rare cells, fine needle aspiration biopsies, and tissue substructures. Herein, we developed an ultrasensitive top-down platform by incorporating a microfluidic sample preparation system, termed nanoPOTS (nanodroplet processing in one pot for trace samples), into a top-down proteomic workflow. A unique combination of a nonionic detergent dodecyl-β-d-maltopyranoside (DDM) with urea as protein extraction buffer significantly improved both protein extraction efficiency and sample recovery. We hypothesize that the DDM detergent improves protein recovery by efficiently reducing nonspecific adsorption of intact proteins on container surfaces, while urea serves as a strong denaturant to disrupt noncovalent complexes and release intact proteins for downstream analysis. The nanoPOTS-based top-down platform reproducibly and quantitatively identified ∼170 to ∼620 proteoforms from ∼70 to ∼770 HeLa cells containing ∼10 to ∼115 ng of total protein. A variety of post-translational modifications including acetylation, myristoylation, and iron binding were identified using only less than 800 cells. We anticipate the nanoPOTS top-down proteomics platform will be broadly applicable in biomedical research, particularly where clinical specimens are not available in amounts amenable to standard workflows.

    Copyright © 2020 American Chemical Society

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    Analytical Chemistry

    Cite this: Anal. Chem. 2020, 92, 10, 7087–7095
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.analchem.0c00467
    Published May 6, 2020
    Copyright © 2020 American Chemical Society
    Request reuse permissions

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