Benchtop-compatible sample processing workflow for proteome profiling of < 100 mammalian cells

Xu, Kerui; Liang, Yiran; Piehowski, Paul D.; Dou, Maowei; Schwarz, Kaitlynn C.; Zhao, Rui; Sontag, Ryan L.; Moore, Ronald J.; Zhu, Ying; Kelly, Ryan T.

doi:10.1007/s00216-018-1493-9

Title: Benchtop-compatible sample processing workflow for proteome profiling of < 100 mammalian cells

Journal Article · 19 November 2018 · Analytical and Bioanalytical Chemistry

DOI: https://doi.org/10.1007/s00216-018-1493-9 · OSTI ID:1701722

Xu, Kerui ^[1]; Liang, Yiran ^[2];

^[3]; Dou, Maowei ^[1]; Schwarz, Kaitlynn C. ^[1]; Zhao, Rui ^[1]; Sontag, Ryan L. ^[3]; Moore, Ronald J. ^[3];

^[1];

^[4]

Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
Brigham Young Univ., Provo, UT (United States)
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL); Brigham Young Univ., Provo, UT (United States)

Extending proteomics to smaller samples can enable the mapping of protein expression across tissues with high spatial resolution and can reveal sub-group heterogeneity. However, despite the continually improving sensitivity of LC-MS instrumentation, in-depth profiling of samples containing low-nanogram amounts of protein has remained challenging due to analyte losses incurred during preparation and analysis. To address this, we recently developed nanodroplet processing in one pot for trace samples (nanoPOTS), a robotic/microfluidic platform that generates ready-to-analyze peptides from cellular material in ~200 nL droplets with greatly reduced sample losses. In combination with ultrasensitive LC-MS, nanoPOTS has enabled >3000 proteins to be confidently identified from as few as 10 cultured human cells and ~700 proteins from single cells. However, the nanoPOTS platform requires a highly skilled operator and a costly in-house-built robotic nanopipetting instrument. In this work, we sought to evaluate the extent to which the benefits of nanodroplet processing could be preserved when upscaling reagent dispensing volumes by a factor of 10 to those addressable by commercial micropipette. We characterized the resulting platform, termed microdroplet processing in one pot for trace samples (µPOTS), for the analysis of as few as ~25 cultured HeLa cells (4 ng total protein) or 50 µm square mouse liver tissue thin sections and found that ~1800 and ~1200 unique proteins were respectively identified with high reproducibility. As such, the reduced equipment requirements should facilitate broad dissemination of nanoproteomics workflows by obviating the need for a capital-intensive custom liquid handling system.

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

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

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 1701722

Report Number(s):: PNNL-SA-146641

Journal Information:: Analytical and Bioanalytical Chemistry, Journal Name: Analytical and Bioanalytical Chemistry Journal Issue: 19 Vol. 411; ISSN 1618-2642

Publisher:: SpringerCopyright Statement

Country of Publication:: United States

Language:: English

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
microfluidics
proteomics
small sample
thin tissue sections

Title: Benchtop-compatible sample processing workflow for proteome profiling of < 100 mammalian cells

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References (48)

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