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

  • Kerui Xu
  • Yiran Liang
  • Paul D. Piehowski
  • Maowei Dou
  • Kaitlynn C. Schwarz
  • Rui Zhao
  • Ryan L. Sontag
  • Ronald J. Moore
  • Ying ZhuEmail author
  • Ryan T. KellyEmail author
Paper in Forefront
Part of the following topical collections:
  1. Young Investigators in (Bio-)Analytical Chemistry

Abstract

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. The reduced equipment requirements should facilitate broad dissemination of nanoproteomics workflows by obviating the need for a capital-intensive custom liquid handling system.

Keywords

Proteomics Small sample Microfluidics Thin tissue sections 

Notes

Acknowledgements

This work was supported by the NIH grants R21 EB020976 and R33 CA225248. This research was performed using EMSL, a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research and located at PNNL.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_1493_MOESM1_ESM.pdf (302 kb)
ESM 1 (PDF 302 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.W.R. Wiley Environmental Molecular Sciences LaboratoryPacific Northwest National LaboratoryRichlandUSA
  2. 2.Department of Chemistry and BiochemistryBrigham Young UniversityProvoUSA
  3. 3.Biological Sciences DivisionPacific Northwest National LaboratoryRichlandUSA

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