Robotic workstation to enable multiplexing for single-cell proteomics

在过去的十年里，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、体内平衡和疾病的认识。核苷酸测序SC方法一直处于该领域的前沿，但在过去的几年里，基于SC质谱的方法已经被开发出来，可以量化数百个细胞中超过一千种蛋白质的表达水平。这允许第一次产生生物学上有意义的，公正的洞察蛋白质组在SC水平。尽管有这些进展，SC蛋白质组学领域仍处于起步阶段，必须克服几个障碍才能使其广泛应用。SC蛋白质组学方法已经变得更加强大，但吞吐量仍然非常有限。使用超灵敏质谱仪和纳米液相色谱（nLC）的无标签方法目前每天可以分析40个细胞。与SC测序方法类似，多路复用是在给定时间内增加可分析细胞数量的一种方法。同位素标记是一种完善的方法，以提高吞吐量多路复用用于质谱为基础的蛋白质组学。通常，胺基用反应性、同位素可区分的化学物质标记，包括甲醛、相对和绝对定量质量差标签（mTRAQ）或串联质量标签（TMT）。最近，通过多路数据独立采集（plexDIA），使用mTRAQ进行多路复用被证明可以将sc蛋白质组学的吞吐量提高3倍。其中一个挑战是，标记低，亚纳克量的肽通常需要在低纳升体积的反应中进行，这在没有专业设备的情况下很难实现。我们最近获得了MRC设备拨款，用于购买与evossep 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来分离、处理和标记SC，以便在纳升反应体积内进行蛋白质组学分析。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品，包括来自bbsrc资助的研究人员的样品，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鲁棒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阐明在衰老、损伤和其他细胞应激和疾病背景下，蛋白质动力学是如何被调节的。重要的是，我们希望探究细胞水平的变化如何影响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.