Integrated Proteome Analysis Device for Fast Single-Cell Protein Profiling
Integrated Proteome Analysis Device for Fast Single-Cell Protein Profiling
复制标题
用于快速单细胞蛋白质分析的集成蛋白质组分析装置
DOI：
10.1021/acs.analchem.8b03692
复制
发表时间：
2018-12-04
影响因子：
7.4
通讯作者：
Zhang, Xiangmin
中科院分区：
文献类型：
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作者：
Shao, Xi;Wang, Xuantang;Zhang, Xiangmin
在我们之前的工作中，我们已经展示了一个集成的蛋白质组分析设备(iPad-100)来分析100个细胞的蛋白质组。(1)在本工作中，首次开发了一种新型的集成单细胞分析设备(iPad-1)，可以在1小时内分析单个细胞中的蛋白质。毛细管。然后在毛细管中以2nL的体积同时完成细胞裂解和蛋白质消化，这可以防止蛋白质的损失和过度稀释。采用高温超声法加速消化。整个细胞处理时间为30min。之后，单细胞消化肽通过真正的零死体积连接直接转移到LC柱中，以最大限度地减少蛋白质转移损失。一架自制的22亩的MID。纳米LC填充柱，内径3微米。该装置使用了ESI探针实现了超灵敏的检测。用30min洗脱程序对单细胞蛋白质组进行分析。在对10个单个HeLa细胞的分析中，使用Orbitrap Fusion Tribrid MS仪器在一个细胞中最多鉴定出328个蛋白质，检测下限估计在1.7-170 zmol左右。IPad-1的灵敏度相当于我们之前开发的iPad-100系统的120倍。(1)在蛋白质表达谱中观察到显著的细胞异质性。此外，我们根据核心组蛋白的数量粗略地估计了被测试的HeLa细胞的细胞周期的各个阶段。
In our previous work, we have demonstrated an integrated proteome analysis device (iPAD-100) to analyze proteomes from 100 cells.(1) In this work, for the first time, a novel integrated device for single-cell analysis (iPAD-1) was developed to profile proteins in a single cell within 1 h. In the iPAD-1, a selected single cell was directly sucked into a 22 mu m i.d. capillary. Then the cell lysis and protein digestion were simultaneously accomplished in the capillary in a 2 nL volume, which could prevent protein loss and excessive dilution. Digestion was accelerated by using elevated temperature with ultrasonication. The whole time of cell treatment was 30 min. After that, single-cell digest peptides were transferred into an LC column directly through a true zero dead volume union, to minimize protein transfer loss. A homemade 22 mu m i.d. nano-LC packing column with 3 mu m i.d. ESI tip was used in the device to achieve ultrasensitive detection. A 30 min elution program was applied to analysis of the single-cell proteome. Therefore, the total time needed for a single-cell analysis was only 1 h. In an analysis of 10 single HeLa cells, a maximum of 328 proteins were identified in one cell by using an Orbitrap Fusion Tribrid MS instrument, and the detection limit was estimated at around 1.7-170 zmol. Such a sensitivity of the iPAD-1 was, similar to 120-fold higher than that of our previously developed iPAD-100 system.(1) Prominent cellular heterogeneity in protein expressive profiling was observed. Furthermore, we roughly estimated the phases of the cell cycle of tested HeLa cells by the amount of core histone proteins.