用于无标记单细胞蛋白质组学的高效、灵敏的样品制备、分离和数据采集。

Q4 Biochemistry, Genetics and Molecular Biology Methods in molecular biology Pub Date : 2024-01-01 DOI:10.1007/978-1-0716-3934-4_7
Thy Truong, Ximena Sanchez-Avila, Kei G I Webber, S Madisyn Johnston, Ryan T Kelly
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引用次数: 0

摘要

我们介绍了一种灵敏高效的无标记单细胞蛋白质组学工作流程,该流程横跨样品制备、液相色谱分离和质谱数据采集。Tecan Uno 单细胞分装仪可在 384 孔 PCR 板内快速进行细胞分离和纳升容量试剂分装。新开发的样品处理工作流程只需一个试剂分配步骤,即可在 1 小时内完成细胞裂解、蛋白质变性和消化。低流量液相色谱法与宽窗口数据采集相结合,使用 Orbitrap Exploris 480 质谱仪对每个细胞的近 3000 种蛋白质进行定量。这种方法大大拓宽了非专业实验室进行灵敏单细胞蛋白质组分析的途径。
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Efficient and Sensitive Sample Preparation, Separations, and Data Acquisition for Label-Free Single-Cell Proteomics.

We describe a sensitive and efficient workflow for label-free single-cell proteomics that spans sample preparation, liquid chromatography separations, and mass spectrometry data acquisition. The Tecan Uno Single Cell Dispenser provides rapid cell isolation and nanoliter-volume reagent dispensing within 384-well PCR plates. A newly developed sample processing workflow achieves cell lysis, protein denaturation, and digestion in 1 h with a single reagent dispensing step. Low-flow liquid chromatography coupled with wide-window data-dependent acquisition results in the quantification of nearly 3000 proteins per cell using an Orbitrap Exploris 480 mass spectrometer. This approach greatly broadens accessibility to sensitive single-cell proteome profiling for nonspecialist laboratories.

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来源期刊
Methods in molecular biology
Methods in molecular biology Biochemistry, Genetics and Molecular Biology-Genetics
CiteScore
2.00
自引率
0.00%
发文量
3536
期刊介绍: For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.
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