Fully Automated Sample Processing and Analysis Workflow for Low-Input Proteome Profiling.
Fully Automated Sample Processing and Analysis Workflow for Low-Input Proteome Profiling.
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DOI：
10.1021/acs.analchem.0c04240
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发表时间：
2021-01-26
影响因子：
7.4
通讯作者：
Kelly RT
中科院分区：
文献类型：
--
作者：
Liang Y;Acor H;McCown MA;Nwosu AJ;Boekweg H;Axtell NB;Truong T;Cong Y;Payne SH;Kelly RT
样品制备和分析的最新进展使得能够直接分析单个哺乳动物细胞和其他痕量样品中的蛋白质表达。制备和分析低输入样品的几种技术采用定制射流技术进行纳升样品处理和手动样品注射到专用分离柱上。虽然有效，但这些高度专业化的系统需要大量的专业知识来制造和操作，这极大地限制了大多数蛋白质组学实验室的实施。在这里，我们报告了一个全自动化的平台，称为autoPOTS（自动制备在一锅微量样品），只使用市售的仪器进行样品处理和分析。使用未经修改的低成本商业机器人移液平台进行一锅法样品制备。我们使用低容量384孔板，并定期向微孔中加入水或缓冲液，以补偿样品孵育期间有限的蒸发。使用商业自动进样器直接从孔板中分析制备的样品，该自动进样器经10端口阀改良，与30 μm i.d.兼容。nanoLC柱。我们使用autoPOTS分析了1-500个HeLa细胞，与我们以前开发的nanoPOTS平台相比，150个细胞的肽覆盖率仅适度降低，单细胞的覆盖率降低了24%。为了评估临床可行性，我们从约130个分选的B或T淋巴细胞中鉴定出平均1095个蛋白质组。我们预计，autoPOTS的直接实现将使其成为许多实验室中低输入和单细胞蛋白质组学的有吸引力的选择。
Recent advances in sample preparation and analysis have enabled direct profiling of protein expression in single mammalian cells and other trace samples. Several techniques to prepare and analyze low-input samples employ custom fluidics for nanoliter sample processing and manual sample injection onto a specialized separation column. While being effective, these highly specialized systems require significant expertise to fabricate and operate, which has greatly limited implementation in most proteomic laboratories. Here, we report a fully automated platform termed autoPOTS (automated preparation in one pot for trace samples) that uses only commercially available instrumentation for sample processing and analysis. An unmodified, low-cost commercial robotic pipetting platform was utilized for one-pot sample preparation. We used low-volume 384-well plates and periodically added water or buffer to the microwells to compensate for limited evaporation during sample incubation. Prepared samples were analyzed directly from the well plate with a commercial autosampler that was modified with a 10-port valve for compatibility with 30 μm i.d. nanoLC columns. We used autoPOTS to analyze 1–500 HeLa cells and observed only a moderate reduction in peptide coverage for 150 cells and a 24% reduction in coverage for single cells compared to our previously developed nanoPOTS platform. To evaluate clinical feasibility, we identified an average of 1095 protein groups from ~130 sorted B or T lymphocytes. We anticipate that the straightforward implementation of autoPOTS will make it an attractive option for low-input and single-cell proteomics in many laboratories.
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影响因子：
9.9
作者：
通讯作者：
--
影响因子：
14.9
作者：
Kuleshov MV;Jones MR;Rouillard AD;Fernandez NF;Duan Q;Wang Z;Koplev S;Jenkins SL;Jagodnik KM;Lachmann A;McDermott MG;Monteiro CD;Gundersen GW;Ma'ayan A
通讯作者：
Ma'ayan A
影响因子：
7
作者：
Li, Siyang;Plouffe, Brian D.;Ivanov, Alexander R.
通讯作者：
Ivanov, Alexander R.
影响因子：
12.3
作者：
Budnik B;Levy E;Harmange G;Slavov N
通讯作者：
Slavov N
DOI：
10.1083/jcb.121.5.1121
发表时间：
1993-06
期刊：
The Journal of cell biology
影响因子：
--
作者：
Bubien JK;Zhou LJ;Bell PD;Frizzell RA;Tedder TF
通讯作者：
Tedder TF