Robotic workstation to enable multiplexing for single-cell proteomics

机器人工作站可实现单细胞蛋白质组学的多重分析

• 批准号: BB/X019160/1
• 负责人: Alex Von Kriegsheim
• 金额: $ 37.54万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX019160%2F1
• 关键词: Robotic; workstation; enable; multiplexing; single

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.

在过去的十年中，单细胞（SC）研究彻底改变了我们对细胞异质性如何调节发育、稳态和疾病的认识。核苷酸测序 SC 方法一直处于该领域的前沿，但在过去几年中，基于 SC 质谱的方法已经开发出来，可以量化数百个细胞中一千多种蛋白质形式的蛋白质表达水平。这使得首次能够在 SC 水平上对蛋白质组产生具有生物学意义的、公正的见解。尽管取得了这些进展，SC 蛋白质组学领域仍处于起步阶段，必须克服一些障碍才能使其广泛应用。 SC 蛋白质组学方法变得更加稳健，但通量仍然非常有限。使用超灵敏质谱仪和纳米液相色谱 (nLC) 的无标记方法目前每天能够分析 40 个细胞。与 SC 测序方法类似，多重测序是增加给定时间范围内可分析的细胞数量的一种方法。同位素标记是一种行之有效的方法，可通过基于质谱的蛋白质组学中的多重分析来提高通量。通常，胺基团用反应性、同位素可区分的化学物质标记，包括甲醛、用于相对和绝对定量的质量差异标签 (mTRAQ) 或串联质量标签 (TMT)。最近，使用 mTRAQ 的多重分析被证明可以通过多重数据独立采集 (plexDIA) 将 SC 蛋白质组通量提高 3 倍。挑战之一是标记低量、亚纳克量的肽通常需要在低纳升体积下进行反应，如果没有专业设备，这可能很难实现。我们最近获得了 MRC 设备拨款，用于购买与 Evosep nLC 系统相结合的 timsTOF 单细胞蛋白质组学 (SCP)。在这里，我们建议购买一个机器人工作站来分离、处理和标记 SC，以便在纳升反应体积中进行蛋白质组分析。该仪器将使我们能够有效地标记 SC 以进行蛋白质组分析，从而实现多重分析并将能力提高至少三倍。增加的通量将用于处理更多样本，包括来自 BBSRC 资助的研究人员的样本，其中一些研究人员提出了如何将 SC 蛋白质组学应用于其工作的项目。除了提高通量外，CellenONE 上的 SC 样品制备还受益于提高稳健性和灵敏度的工作流程。这是 CellenONE 仪器迅速成为 SC 蛋白质组学样品制备标准的关键原因之一。该系统最初将由来自爱丁堡各地的研究人员组成的联盟使用，以研究植物、动物、寄生虫和人类的细胞异质性。我们正在进行的工作将揭示衰老、损伤以及其他细胞应激和疾病背景下蛋白质动力学如何受到调节。重要的是，我们希望探究细胞水平的变化如何影响组织或有机体对环境挑战的反应。