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Profiling embryonic stem cell differentiation by MALDI TOF mass spectrometry: development of a reproducible and robust sample preparation workflow

Rachel E. Heap, Anna Segarra Fas, Alasdair P. Blain, Greg M. Findlay, Matthias Trost
doi: https://doi.org/10.1101/536664
Rachel E. Heap
1Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle-upon-Tyne, UK
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Anna Segarra Fas
2MRC Protein Phosphorylation & Ubiquitylation Unit, University of Dundee, Dundee, Scotland, UK
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Alasdair P. Blain
3Wellcome Trust Centre for Mitochondrial Research, Newcastle University, Newcastle-upon-Tyne, UK
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Greg M. Findlay
2MRC Protein Phosphorylation & Ubiquitylation Unit, University of Dundee, Dundee, Scotland, UK
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Matthias Trost
1Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle-upon-Tyne, UK
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  • For correspondence: matthias.trost@ncl.ac.uk
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Abstract

MALDI TOF mass spectrometry (MS) is widely used to characterize and biotype bacterial samples, but a complimentary method for profiling of mammalian cells is still underdeveloped. Current approaches vary dramatically in their sample preparation methods and are not suitable for high-throughput studies. In this work, we present a universal workflow for mammalian cell MALDI TOF MS analysis and apply it to distinguish ground-state naïve and differentiating mouse embryonic stem cells (mESCs), which can be used as a model for drug discovery. We employed a systematic approach testing many parameters to evaluate how efficiently and reproducibly each method extracted unique mass features from four different human cell lines. This data enabled us to develop a unique mammalian cell MALDI TOF workflow involving a freeze-thaw cycle, methanol fixing and CHCA matrix to generate spectra that robustly phenotype different cell lines and are highly reproducible in peak identification across replicate spectra. We applied our optimised workflow to distinguish naïve and differentiating populations using multivariate analysis and reproducibly identifying unique features. We were also able to demonstrate the compatibility of our optimised method for current automated liquid handling technologies. Consequently, our MALDI TOF MS profiling method enables identification of unique features and robust phenotyping of mESC differentiation in under 1 hour from culture to analysis, which is significantly faster and cheaper when compared with conventional methods such as qPCR. This method has the potential to be automated and can in the future be applied to profile other cell types and expanded towards cellular MALDI TOF MS screening assays.

  • MALDI TOF
  • mass spectrometry
  • stem cells
  • whole-cell profiling
  • phenotypic screen
  • drug discovery

Footnotes

  • Mailing address: M.T.: Newcastle University, Institute for Cell and Molecular Biosciences, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, Phone: +44 191 2087009

  • Some improvements

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted July 09, 2019.
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Profiling embryonic stem cell differentiation by MALDI TOF mass spectrometry: development of a reproducible and robust sample preparation workflow
Rachel E. Heap, Anna Segarra Fas, Alasdair P. Blain, Greg M. Findlay, Matthias Trost
bioRxiv 536664; doi: https://doi.org/10.1101/536664
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Profiling embryonic stem cell differentiation by MALDI TOF mass spectrometry: development of a reproducible and robust sample preparation workflow
Rachel E. Heap, Anna Segarra Fas, Alasdair P. Blain, Greg M. Findlay, Matthias Trost
bioRxiv 536664; doi: https://doi.org/10.1101/536664

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