A cellenONE BSC to enhance UK capability for single-cell proteomics

Single-cell studies are transforming many areas of biological research; these include studies of infection, differentiation, construction of cell atlases, and more. Single-cell proteomics is a rapidly emerging addition to the single-cell toolset, with single-cell proteomics by mass spectrometry capable of measuring the abundance of thousands of proteins across hundreds to thousands of single-cells. By acquiring state-of-the-art instrumentation for single-cell proteomics sample preparation, this proposal seeks to enhance UK capabilities in single-cell proteomics. Specific applications of this instrument include:
i) Further development and application of single-cell proteomics. The cellenONE increases the sensitivity of single-cell proteomics by minimizing sample losses during processing. We will exploit this increased sensitivity to target specific proteins of interest for reproducible analysis across single-cells, study post-translational modifications at single-cell level, as well as apply this system to develop new methods for spatial and subcellular single-cell 'omics.
ii) Cell/picolitre dispensing for other single-cell approaches. In this proposal we specifically apply this instrument to single-cell western blotting, providing information on protein molecular weight, and more sensitive comparative analysis as the direct introduction of cells into nanowells permits multiple samples to be analysed per chip, minimizing batch effects. Similarly, the cellenONE instrument will be applied to single-cell RNAseq studies, where the gentle acoustic dispensing used by the system can overcome cell-size-based biases or problems analysing fragile cells in existing approaches.

We propose to bring this novel instrumentation which enables automation of the entire single-cell proteomics workflow to the Centre for Proteome Research (CPR) at the University of Liverpool (UoL). Making full use of our existing suite of LC-MS/MS instruments, and using our established cost recovery model which ensures long-term maintenance and support (supported by LIV-SRF which oversees research facilities at UoL), this instrumentation will be made available to researchers working across BBSRC priority areas, both locally and nationally. LIV-SRF provides financial support to maintain and develop open facilities such as the CPR, including long-term support of staff. LIV-SRF also awards 'LIV-SRF Vouchers' which provide academics, especially ECRs with matched funds to support facility usage and develop preliminary data, to support future funding or fellowship applications.

All five current cellenONE systems in academic labs in the UK are located in London or the East of England, and none are currently used for single-cell proteomics. This instrument will be applied to multiple BBSRC-funded research areas, and our demonstration projects and letters of support for this system have focused on research impacting infection biology, fundamental cell signaling research, and immunity. This instrument will enable us to both directly contribute to these research areas, as well as support training for other UK academics and industry users to establish single-cell proteomics at their own institutions.

We propose to install a state-of-the art cellenONE F1.4 BSC cell dispensing and picolitre liquid handling instrument in the Centre for Proteome Research (CPR), University of Liverpool. This acquisition will enhance CPR and UK capabilities in single-cell proteomics, and more broadly in single-cell biology. The cellenONE represents the sole commercially-available instrument to enable end-to-end sample processing of single-cell proteomics samples for mass spectrometry, and benefits from the instruments 'open' format, with methods and consumables freely definable in the software. The cellenONE F1.4 BSC is especially suitable for this research as it permits:
i) Sorting and gating of single-cells based on bright-field and fluorescence parameters, providing pre- and post-sorting micrographs of individual cells
ii) Picolitre liquid handling: cellenONE-based workflows for single-cell proteomics have diminished sample processing volumes 100-fold, from 1-2 microlitres to <20 nL. Lower sample volumes result in significantly reduced sample losses during processing.
iii) Temperature, humidity and dew point control, permitting all steps of sample preparation (sorting, digestion, sample labeling) to all occur within the cellenONE, without requiring additional sample handling.
iv) Open consumables format: Options for single-cell proteomics include the newly-released proteoCHIP, specifically designed for the cellenONE, and sample preparation on fluorocarbon-treated slides.

The gains in sensitivity (limits of detection), open platform, and now commercial single-cell proteomic sample preparation solutions (proteoCHIP) are key reasons why the international single-cell proteomics community has rapidly adopted the cellenONE instrument. The acquisition of a cellenONE instrument by the CPR is crucial if we are to maintain UK proteomics research at the forefront of this rapidly evolving field, with applications across fundamental biological and biomedical research.