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基于质谱的单细胞蛋白质组学分析方法及应用

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 he...

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Detalles Bibliográficos
Autores principales: QIN, Shaojie, BAI, Yu, LIU, Huwei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Editorial board of Chinese Journal of Chromatography 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9274836/
https://www.ncbi.nlm.nih.gov/pubmed/34227347
http://dx.doi.org/10.3724/SP.J.1123.2020.08030
Descripción
Sumario: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.