Laser capture microdissection-capillary zone electrophoresis-tandem mass spectrometry (LCM-CZE-MS/MS) for spatially resolved top-down proteomics: a pilot study of zebrafish brain.
Laser capture microdissection-capillary zone electrophoresis-tandem mass spectrometry (LCM-CZE-MS/MS) for spatially resolved top-down proteomics: a pilot study of zebrafish brain.
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激光捕获显微切割-毛细管区带电泳-串联质谱（LCM-CZE-MS/MS）用于空间分辨自上而下蛋白质组学：斑马鱼脑的初步研究。
DOI：
10.1039/d1mo00335f
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发表时间：
2022-02-21
期刊：
影响因子：
2.9
通讯作者：
中科院分区：
文献类型：
--
作者：
基于质谱（MS）的组织空间分辨自上而下蛋白质组学（TDP）对于理解器官生物学功能中微环境异质性所起的作用以及发现新的疾病蛋白质标志物至关重要。很少有已发表的空间分辨TDP研究。挑战之一涉及TDP用于使用例如激光捕获显微切割（LCM）分析空间隔离的样品的有限性能，因为那些样品通常是质量受限的。本文首次以斑马鱼脑为样品，对液相色谱-毛细管区带电泳-串联质谱法（LCM-CZE-MS/MS）进行了空间分辨TDP的初步研究。LCM-CZE-MS/MS平台采用非离子型去污剂和冻融方法从LCM分离的脑切片中进行有效的蛋白质型提取，然后进行CZE-MS/MS，无需任何样品净化步骤，确保高灵敏度。通过消耗大约250个细胞的蛋白质含量，在一个LCM脑切片的CZE-MS/MS分析中鉴定了超过400种蛋白质型。我们观察到两个LCM脑切片分离的视顶盖（Teo）和端脑（Tel）地区之间的蛋白质型配置文件的巨大差异。具有神经肽激素活性的三种蛋白质（npy、penkb和pyya）的蛋白质形式在分离的Tel部分中被专门鉴定。蛋白质的reticulon，肌球蛋白，肌钙蛋白几乎完全确定在孤立的Teo部分，这些蛋白质在视觉和运动活动中发挥重要作用。蛋白质型谱准确地反映了大脑Teo和Tel区域的主要生物学功能。此外，还鉴定了数百种后修饰的蛋白质型。
Mass spectrometry (MS)-based spatially resolved top-down proteomics (TDP) of tissues is crucial for understanding the roles played by microenvironmental heterogeneity in the biological functions of organs and for discovering new proteoform biomarkers of diseases. There are few published spatially resolved TDP studies. One of the challenges relates to the limited performance of TDP for the analysis of spatially isolated samples using, for example, laser capture microdissection (LCM) because those samples are usually mass-limited. We present the first pilot study of LCM-capillary zone electrophoresis (CZE)-MS/MS for spatially resolved TDP and used zebrafish brain as the sample. The LCM-CZE-MS/MS platform employed a non-ionic detergent and a freeze–thaw method for efficient proteoform extraction from LCM isolated brain sections followed by CZE-MS/MS without any sample cleanup step, ensuring high sensitivity. Over 400 proteoforms were identified in a CZE-MS/MS analysis of one LCM brain section via consuming the protein content of roughly 250 cells. We observed drastic differences in proteoform profiles between two LCM brain sections isolated from the optic tectum (Teo) and telencephalon (Tel) regions. Proteoforms of three proteins (npy, penkb, and pyya) having neuropeptide hormone activity were exclusively identified in the isolated Tel section. Proteoforms of reticulon, myosin, and troponin were almost exclusively identified in the isolated Teo section, and those proteins play essential roles in visual and motor activities. The proteoform profiles accurately reflected the main biological functions of the Teo and Tel regions of the brain. Additionally, hundreds of post-translationally modified proteoforms were identified.
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影响因子：
7.4
作者：
Lubeckyj RA;McCool EN;Shen X;Kou Q;Liu X;Sun L
通讯作者：
Sun L
影响因子：
64.8
作者：
Konig, M;Zimmer, AM;Zimmer, A
通讯作者：
Zimmer, A
影响因子：
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作者：
Kou, Qiang;Xun, Likun;Liu, Xiaowen
通讯作者：
Liu, Xiaowen
影响因子：
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作者：
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通讯作者：
Fischer, Roman
DOI：
10.1007/s13361-019-02167-w
发表时间：
2019-08-01
影响因子：
3.2
作者：
Lubeckyj, Rachele A.;Basharat, Abdul Rehman;Sun, Liangliang
通讯作者：
Sun, Liangliang