CEEPC/IPM/CMSC - Abstrakt prezentace

(CEEPC/IPM/CMSC 2022 - PL-01)
New insights through Single-Cell Proteomics

Claudia Ctortecka 1, David Hartlmayr 1, Manuel Matzinger 1, Elisabeth Müller 1, Anjali Seth 2, Sasha Mendjan 3, Guilhem Tourniaire 2, Karl Mechtler 1,3 *

  1. Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Campus-Vienna-Biocenter 1, 1030 Vienna, Austria
  2. Cellenion SASU, 60F avenue Rockefeller, 69008 Lyon, France
  3. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria

Abstrakt

The analysis of single cell proteomes has recently become a viable complement to transcriptomics 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, optimized for multiplexed single cell proteomics sample preparation at surprising sensitivity and throughput. Sample processing using the cellenONE® robot, allows to reduce final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error prone manual sample handling and overcoming evaporation. This results in around 1,500 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,600 proteins across 170 multiplexed single cells from two highly similar human cell types. This dedicated loss-less workflow allows to distinguish in vitro co-differentiated cell types of self-organizing cardiac organoids based on indicative markers across 150 single cells. In-depth characterization revealed enhanced cellular motility of endothelial cells and acute myocardium sarcomere organization in cardiomyocytes.
In addition, we evolved a robust and sensitive one-pot label free single cell workflow. By working in standard 384 well plates and compatibility with both, cell sorting in the cellenONE® or using alternatives like a FACS device the need for specialized equipment is obsolete making this workflow easy to use, cheap and accessible to a broader community. By keeping the sample in a hydrated state during proteolytic digestion and addition of DMSO for storage we improved recovery of hydrophobic peptides and boosted ID numbers to more than 1000 proteins from a single cell without match between runs.
In conclusion, our versatile, and semi-automated sample preparation workflows have not only proven to be easily adoptable but are also sufficiently sensitive to drive biological applications of single cell proteomics.

* Korespondující autor: mechtler@imp.ac.at


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