Robotic workstation to enable multiplexing for single-cell proteomics

在过去的十年中，单细胞（SC）研究已经彻底改变了我们对细胞异质性如何调节发育，稳态和疾病的见解。核苷酸测序SC方法一直处于该领域的前沿，但在过去的几年中，已经开发了基于SC质谱的方法，可以量化数百个细胞中超过一千种蛋白质型的蛋白质表达水平。这使得第一次在SC水平上对蛋白质组产生生物学上有意义的，无偏见的见解。尽管有这些进展，SC蛋白质组学领域仍处于起步阶段，必须克服几个障碍，使其广泛适用。SC蛋白质组学方法已经变得更加稳健，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。类似于已经开发的SC测序方法，多路复用是增加可以在给定时间范围内分析的细胞数量的一种方法。同位素标记是通过在基于质谱的蛋白质组学中使用的多路复用来增加通量的成熟方法。通常，胺基团用反应性的同位素可区分的化学品标记，包括甲醛、用于相对和绝对定量的质量差标签（mTRAQ）或串联质量标签（TMT）。最近，使用mTRAQ的多路复用被证明通过多路复用数据独立采集（plexDIA）将SC-蛋白质组学通量增加3倍。挑战之一是，低、亚纳克量肽的标记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设备资助，用于购买与Evosep nLC系统耦合的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分离，处理和标记SC蛋白质组学分析纳升反应体积。该仪器将使我们能够有效地标记SC进行蛋白质组学分析，从而实现多路复用并将容量提高至少三倍。增加的通量将用于处理更多的样本，包括来自BBSRC资助的研究人员的样本，其中一些人已经提出了如何将SC蛋白质组学应用于他们的工作的项目。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的一个研究者联盟用于研究植物、动物、寄生虫和人类的细胞异质性。我们正在做的工作将揭示蛋白质动力学如何在衰老，损伤和其他细胞应激和疾病背景下进行调节。重要的是，我们希望询问细胞水平的变化如何塑造组织或生物体对环境挑战的反应。

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.