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Routine Workflow of Spatial Proteomics on Micro-formalin-Fixed Paraffin-Embedded Tissues

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机构: [1]College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China. [2]BGI-Shenzhen, Shenzhen, Guangdong 518083, China. [3]Zhejiang Cancer Hospital, Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China. [4]College of Pharmaceutical Science, Zhejiang University of Technology, Gongda Road 1, Huzhou 313200, China. [5]Shanghai University of Traditional Chinese Medicine, Shanghai 200120, China.
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In the era of single-cell biology, spatial proteomics has emerged as an important frontier. However, it still faces several challenges in technology. Formalin-fixed paraffin-embedded (FFPE) tissues are an important material in spatial proteomics, in which fixed tissues are excised using laser capture microdissection (LCM), followed by protein identification with mass spectrometry. For a satisfied spatial proteomics upon FFPE tissues, the excision area is expected to be as small as possible, and the identified proteins are countered upon as much as possible. For a general laboratory for spatial proteomics, a routine workflow is required, not relying on any special device, and is easily operating. In view of these challenges in technology, we initiated a technology evaluation throughout the entire procedure of proteomic analysis with micro-FFPE tissues. In contrast to the protocols reported previously, several innovations in technology were proposed and conducted, such as removal of destaining, decross-linking with "hang-down", solution simplification for peptide generation and balancing to excision area, and capture rate of micro-FFPE tissues. After optimization of all the necessary steps, a routine workflow was established, in which the minimized area for protein identification was 0.002 mm2, while the excision area for a consistent proteomic analysis was 0.05 mm2. Using the developed workflow and collecting the micro-FFPE tissues continuously, for the first time, a spatial proteomic atlas of mouse brain was preliminarily constructed, which exhibited the typical characteristics of spatial-dependent protein abundance and functional enrichment.

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出版当年[2022]版:
大类 | 1 区 化学
小类 | 1 区 分析化学
最新[2025]版:
大类 | 1 区 化学
小类 | 1 区 分析化学
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第一作者机构: [1]College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China. [2]BGI-Shenzhen, Shenzhen, Guangdong 518083, China. [3]Zhejiang Cancer Hospital, Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China.
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通讯机构: [1]College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China. [2]BGI-Shenzhen, Shenzhen, Guangdong 518083, China. [3]Zhejiang Cancer Hospital, Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China.
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