Robotic workstation to enable multiplexing for single-cell proteomics
Lead Research Organisation:
University of Edinburgh
Department Name: Edinburgh Cancer Research Centre
Abstract
Over the past ten years, single-cell (SC) studies have revolutionised our insight into how cellular heterogeneity regulates development, homeostasis and disease. Nucleotide-sequencing SC methods have been at the vanguard of the field, but over the past few years, SC mass spectrometry-based methods have been developed that can quantify protein expression levels for over a thousand proteoforms across hundreds of cells. This allows for the first time to generate biologically meaningful, unbiased insight into the proteome at the SC level.
Despite these developments, the field of SC proteomics is still in its infancy and several hurdles must be overcome to make it broadly applicable. SC proteomics methods have become more robust, but throughput is still very limited. Label-free methods using ultra-sensitive mass spectrometers and nano-liquid chromatography (nLC) are currently able to analyse 40 cells per day. Analogous to what has been developed for SC sequencing methods, multiplexing is one way to increase the number of cells that can be analysed in a given time frame. Isotopic labelling is a well-established method to increase throughput by multiplexing used in mass spectrometry-based proteomics. Commonly, the amine groups are labelled with reactive, isotopically distinguishable chemicals, including formaldehyde, mass-differential Tags for Relative and Absolute Quantitation (mTRAQ) or Tandem Mass Tags (TMT). Recently multiplexing using mTRAQ was demonstrated to increase SC-proteomics throughput 3-fold via multiplexed Data Independent Acquisition (plexDIA). One of the challenges is that labelling of low, sub-nanogram amounts of peptides typically requires the reactions to take place in low nanolitre volumes, which can be difficult to achieve without specialist equipment.
We were recently awarded an MRC equipment grant to purchase a timsTOF Single-cell-proteomics (SCP) coupled to an Evosep nLC system. Here we propose to purchase a robotic workstation to isolate, process and label SC for proteomic analysis in nanolitre reaction volumes. The instrument will permit us to efficiently label SC for proteomic analysis enabling multiplexing and enhancing capacity by at least three-fold. The increased throughput will be used to process additional samples, including those from BBSRC-funded researchers, some of whom have proposed projects on how to apply SC proteomics to their work. In addition to increasing throughput, SC sample preparation on the CellenONE benefits from workflows that increase robustness and sensitivity. This is one of the key reasons why the CellenONE instrument has rapidly become the standard for SC proteomics sample preparation.
The system will be initially used by a consortium of investigators from across Edinburgh to investigate cellular heterogeneity in plants, animals, parasites and humans. The work we are doing will shed light on how protein dynamics are regulated during ageing, injury, and other cellular stress and disease contexts. Importantly, we wish to interrogate how changes at the cellular level shape the response of the tissue or organism to environmental challenges.
Despite these developments, the field of SC proteomics is still in its infancy and several hurdles must be overcome to make it broadly applicable. SC proteomics methods have become more robust, but throughput is still very limited. Label-free methods using ultra-sensitive mass spectrometers and nano-liquid chromatography (nLC) are currently able to analyse 40 cells per day. Analogous to what has been developed for SC sequencing methods, multiplexing is one way to increase the number of cells that can be analysed in a given time frame. Isotopic labelling is a well-established method to increase throughput by multiplexing used in mass spectrometry-based proteomics. Commonly, the amine groups are labelled with reactive, isotopically distinguishable chemicals, including formaldehyde, mass-differential Tags for Relative and Absolute Quantitation (mTRAQ) or Tandem Mass Tags (TMT). Recently multiplexing using mTRAQ was demonstrated to increase SC-proteomics throughput 3-fold via multiplexed Data Independent Acquisition (plexDIA). One of the challenges is that labelling of low, sub-nanogram amounts of peptides typically requires the reactions to take place in low nanolitre volumes, which can be difficult to achieve without specialist equipment.
We were recently awarded an MRC equipment grant to purchase a timsTOF Single-cell-proteomics (SCP) coupled to an Evosep nLC system. Here we propose to purchase a robotic workstation to isolate, process and label SC for proteomic analysis in nanolitre reaction volumes. The instrument will permit us to efficiently label SC for proteomic analysis enabling multiplexing and enhancing capacity by at least three-fold. The increased throughput will be used to process additional samples, including those from BBSRC-funded researchers, some of whom have proposed projects on how to apply SC proteomics to their work. In addition to increasing throughput, SC sample preparation on the CellenONE benefits from workflows that increase robustness and sensitivity. This is one of the key reasons why the CellenONE instrument has rapidly become the standard for SC proteomics sample preparation.
The system will be initially used by a consortium of investigators from across Edinburgh to investigate cellular heterogeneity in plants, animals, parasites and humans. The work we are doing will shed light on how protein dynamics are regulated during ageing, injury, and other cellular stress and disease contexts. Importantly, we wish to interrogate how changes at the cellular level shape the response of the tissue or organism to environmental challenges.
Technical Summary
We have been recently awarded an MRC grant to purchase a timsTOF SCP instrument coupled with an Evosep One nLC that will deliver SC proteomics analyses to research groups in Edinburgh and across Scotland. Due to the unmatched sensitivity, the instrument is the current standard for "true single cell" analysis, but sample throughput is currently limited to 40 cells per day. We propose to introduce isotopic-label multiplexing which will multiply capacity and reduce per-cell sample cost.
Here we propose to purchase a robotic workstation designed to isolate, process and label SC. Effective labelling of low, sub-nanogram amounts of peptides typically requires the reaction to take place in low nanolitre volumes which is not achievable when done manually. The CellenONE workstation (Scienion, France) is a commercial system that has the proven ability to automate the entire workflow from isolation, digestion, labelling, and mixing to loading onto sample plates or Evotips. Processing is done in nanolitre volumes resulting in low sample losses and unparalleled sensitivity. The increased throughput will allow us to collaborate with additional groups, including BBSRC-funded groups in Edinburgh and the Roslin Institute (RI). Such a system is not yet available in Scotland, and when combined with a timsTOF SCP, would be unique in the UK.
We will apply SC proteomics to research questions that would be unapproachable using other methods as the defining processes investigated are regulated by altered proteostasis would this be due to autophagy, proteasomal/lysosomal degradation, Endoplasmic reticulum (ER)-associated protein degradation (ERAD), Ubiquitylation, translation, stabilisation or other. The research topics we are going to investigate span the entire BBSRC remit, including plants, animals, zoonotic pathogens, biotechnology, healthy ageing and understanding the rules of life.
Here we propose to purchase a robotic workstation designed to isolate, process and label SC. Effective labelling of low, sub-nanogram amounts of peptides typically requires the reaction to take place in low nanolitre volumes which is not achievable when done manually. The CellenONE workstation (Scienion, France) is a commercial system that has the proven ability to automate the entire workflow from isolation, digestion, labelling, and mixing to loading onto sample plates or Evotips. Processing is done in nanolitre volumes resulting in low sample losses and unparalleled sensitivity. The increased throughput will allow us to collaborate with additional groups, including BBSRC-funded groups in Edinburgh and the Roslin Institute (RI). Such a system is not yet available in Scotland, and when combined with a timsTOF SCP, would be unique in the UK.
We will apply SC proteomics to research questions that would be unapproachable using other methods as the defining processes investigated are regulated by altered proteostasis would this be due to autophagy, proteasomal/lysosomal degradation, Endoplasmic reticulum (ER)-associated protein degradation (ERAD), Ubiquitylation, translation, stabilisation or other. The research topics we are going to investigate span the entire BBSRC remit, including plants, animals, zoonotic pathogens, biotechnology, healthy ageing and understanding the rules of life.