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An automated workflow for label-free and multiplexed single cell proteomics sample preparation at unprecedented sensitivity

David Hartlmayr, View ORCID ProfileClaudia Ctortecka, View ORCID ProfileAnjali Seth, View ORCID ProfileSasha Mendjan, View ORCID ProfileGuilhem Tourniaire, View ORCID ProfileKarl Mechtler
doi: https://doi.org/10.1101/2021.04.14.439828
David Hartlmayr
1Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Campus-Vienna-Biocenter 1, 1030 Vienna, Austria
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Claudia Ctortecka
1Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Campus-Vienna-Biocenter 1, 1030 Vienna, Austria
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  • For correspondence: Claudia.ctortecka@imp.ac.at Karl.mechtler@imp.ac.at
Anjali Seth
4Cellenion SASU, 60F avenue Rockefeller, 69008 Lyon, France
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Sasha Mendjan
2Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
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Guilhem Tourniaire
4Cellenion SASU, 60F avenue Rockefeller, 69008 Lyon, France
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Karl Mechtler
1Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Campus-Vienna-Biocenter 1, 1030 Vienna, Austria
2Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
3The Gregor Mendel Institute of Molecular Plant Biology of the Austrian Academy of Sciences (GMI), Vienna BioCenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
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  • ORCID record for Karl Mechtler
  • For correspondence: Claudia.ctortecka@imp.ac.at Karl.mechtler@imp.ac.at
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Abstract

The analysis of single cell proteomes has recently become a viable complement to transcript and genomics studies. Proteins are the main driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Both multiplexed and label-free mass spectrometry-based approaches with single cell resolution have lately attributed surprising heterogeneity to believed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lacks behind. Here, we introduce the proteoCHIP, a universal option for single cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single cell isolation and picoliter dispensing, the cellenONE®, allows to reduce final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error prone manual sample handling and overcoming evaporation. With this specialized workflow we achieved around 1,000 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,000 protein groups across 158 multiplexed single cells from two highly similar human cell types and clustered them based on their proteome. In-depth investigation of regulated proteins readily identified one of the main drivers for tumorigenicity in this cell type. Our workflow is compatible with all labeling reagents, can be easily adapted to custom workflows and is a viable option for label-free sample preparation. The specialized proteoCHIP design allows for the direct injection of label-free single cells via a standard autosampler resulting in the recovery of 30% more protein groups compared to samples transferred to PEG coated vials. We therefore are confident that our versatile, sensitive, and automated sample preparation workflow will be easily adoptable by non-specialized groups and will drive biological applications of single cell proteomics.

Competing Interest Statement

A.S. and G.T. are employees of Cellenion.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted April 14, 2021.
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An automated workflow for label-free and multiplexed single cell proteomics sample preparation at unprecedented sensitivity
David Hartlmayr, Claudia Ctortecka, Anjali Seth, Sasha Mendjan, Guilhem Tourniaire, Karl Mechtler
bioRxiv 2021.04.14.439828; doi: https://doi.org/10.1101/2021.04.14.439828
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An automated workflow for label-free and multiplexed single cell proteomics sample preparation at unprecedented sensitivity
David Hartlmayr, Claudia Ctortecka, Anjali Seth, Sasha Mendjan, Guilhem Tourniaire, Karl Mechtler
bioRxiv 2021.04.14.439828; doi: https://doi.org/10.1101/2021.04.14.439828

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