The cell is the smallest unit of living organisms. Although cells often assemble to serve a common function, intercellular heterogeneity often exists due to different genetic and environmental effects. Therefore, single-cell analysis has been regarded as an indispensable means to investigate cell heterogeneity, especially when researching cell differentiation, disease diagnosis, and therapy. As the chief factors influencing cell and biological activities, proteins have long been a major concern in biochemistry. However, due to their intrinsic lack of amplification characteristics, wide species variety, low abundance, and wide dynamic range, proteins are scarcely studied in single-cell research when compared with other biological macromolecules. Therefore, ultra-sensitive single-cell proteomics analysis methods are urgently required. Among all general measurement techniques, fluorescence methods possess high sensitivity and a capability of dynamic tracing, but low target numbers impose restrictions on their broad application in real "proteomic" studies. Similarly, electrochemical methods adapt to electrochemically active molecules, which miss the majority of proteins. Mass spectrometry (MS), as the core approach of proteomic studies, provides high-sensitivity and high-throughput analysis of proteins together with abundant structural information, which is unique in all the analytical instruments and has made great progress in single-cell proteomic research. Herein, the representative research methods for single-cell proteomics based on MS are reviewed. According to the different protein separation methods used prior to MS analysis, they are divided into three categories, including capillary electrophoresis (CE), liquid chromatography (LC), and direct infusion without the need for separation. First, CE has been widely used in the separation and analysis of complex biological samples owing to its low cost, high analysis speed, and high separation efficiency. Its unique feature is the extraction and transfer of contents from cellular or subcellular regions using capillaries smaller than a single cell size. This sampling method also offers less substrate interference and negligible oxidative damage to the cells. Nonetheless, single-cell analysis based on CE-MS mainly focuses on proteomic studies of large cells because of the considerable sample loss, interface instability, and reproducibility issues. Compared with CE, LC, especially nanoLC, is more widely used in single-cell proteomic research, which mainly depends on its good reproducibility, nanoliter injection volume, low flow rate, low sample loss, and good compatibility with mass spectrometry. In recent years, it has been increasingly applied in the study of large-volume embryos, germ cells, and even somatic cells. More than 1000 proteins have been identified in single HeLa cells using this state-of-the-art single-cell proteomics method. It is worth noting that the single-cell sampling volume based on LC gradually reduces to the nanoliter level, and that the sample loss can be reduced by integrating a series of proteomic sampling processes into small volumes, setting sealing conditions, and reducing washing steps. However, the adequacy of cell lysis, the completeness and efficiency of protein pretreatment, and the labeling of peptide segments are important factors affecting the number and types of protein identification. Compared with protein separation using CE or LC prior to MS analysis, the direct MS analysis, assisted by labelling and signal transformation, eliminates complicated sample pretreatment and simplifies the operation by reducing enzymatic hydrolysis and separation. It also renders higher resolution as well as multi-omics compatibility. So far, the number of proteins detected using this method is limited due to the complexity of the samples. In conclusion, the aspects of throughput, sensitivity, identified protein species, and applications are summarized for each method mentioned above, and the prospect of single-cell proteomic research based on MS in the future is also discussed.
细胞是生命体的最小组成单位,遗传及外部环境等因素使单细胞异质性广泛存在于众多生物体中。传统的生物学实验获得的结果多是大量细胞的平均测量值,因此在单细胞层面开展研究对于精确理解细胞的生长发育以及疾病的诊断与治疗至关重要。而作为重要的细胞和生命活动的执行者,蛋白质由于其不具备扩增特性,且种类繁多、丰度低、动态分布范围宽,与核酸等其他生物大分子相比,其单细胞组学研究相对滞后。而在所有的检测手段中,荧光检测以及电化学分析方法具有极高的灵敏度,但是囿于其研究通量有限,以及电化学活性依赖,很难成为普适性的单细胞蛋白质组学研究方法。质谱分析作为传统蛋白质组学中最为核心的研究技术,由于其高灵敏、高通量、结构信息丰富等特点,在单细胞蛋白质组学研究中独树一帜。该文综述了近年来基于质谱的单细胞蛋白质组学研究中的代表性方法,根据质谱分析前蛋白质分离方式的差异,将其分为基于毛细管电泳分离、液相色谱分离和无分离手段的直接检测3类方法,在介绍研究现状的同时对这些方法在细胞通量、蛋白质鉴定数目、灵敏度以及方法应用方面进行了总结与比较。最后,基于目前研究中面临的挑战以及发展趋势对基于质谱的单细胞蛋白质组学的研究前景进行了展望。
Keywords: capillary electrophoresis (CE); liquid chromatography (LC); mass spectrometry (MS); microfluidic; proteomics; review; single cell.