Microsampling Capillary Electrophoresis Mass Spectrometry Enables Single-Cell Proteomics in Complex Tissues: Developing Cell Clones in Live Xenopus laevis and Zebrafish Embryos
Microsampling Capillary Electrophoresis Mass Spectrometry Enables Single-Cell Proteomics in Complex Tissues: Developing Cell Clones in Live Xenopus laevis and Zebrafish Embryos
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DOI：
10.1021/acs.analchem.9b00345
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发表时间：
2019-04-02
影响因子：
7.4
通讯作者：
Nemes, Peter
中科院分区：
文献类型：
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作者：
Lombard-Banek, Camille;Moody, Sally A.;Nemes, Peter
无标记的单细胞蛋白质组质谱(MS)目前与复杂组织不兼容，不需要细胞培养、单细胞解剖或组织分离。我们在这里报告了第一个无标记的基于MS的蛋白质组学的例子，直接在活的脊椎动物胚胎的单细胞中进行。我们的方法包括光学引导的原位亚细胞毛细管微采样，一锅提取-消化收集的蛋白质，毛细管电泳法分离多肽，超灵敏电动泵浦纳米电喷雾电离，以及高分辨率MS(Orbitrap)检测。凭借700 zmol(420,000拷贝)的检测下限，这项跟踪敏感技术可以自信地识别和定量类似于750-800个蛋白质组(
Label-free single-cell proteomics by mass spectrometry (MS) is currently incompatible with complex tissues without requiring cell culturing, single-cell dissection, or tissue dissociation. We here report the first example of label-free single-cell MS-based proteomics directly in single cells in live vertebrate embryos. Our approach integrates optically guided in situ subcellular capillary microsampling, one-pot extraction-digestion of the collected proteins, peptide separation by capillary electrophoresis, ionization by an ultrasensitive electrokinetically pumped nanoelectrospray, and detection by high-resolution MS (Orbitrap). With a 700 zmol (420 000 copies) lower limit of detection, this trace-sensitive technology confidently identified and quantified similar to 750-800 protein groups (