High-throughput proteomic sample preparation using pressure cycling technology.
High-throughput proteomic sample preparation using pressure cycling technology.
复制标题
使用压力循环技术制备高通量蛋白质组样品
DOI：
10.1038/s41596-022-00727-1
复制
发表时间：
2022-10
期刊：
影响因子：
14.8
通讯作者：
Guo, Tiannan
中科院分区：
文献类型：
--
作者：
Cai, Xue;Xue, Zhangzhi;Wu, Chunlong;Sun, Rui;Qian, Liujia;Yue, Liang;Ge, Weigang;Yi, Xiao;Liu, Wei;Chen, Chen;Gao, Huanhuan;Yu, Jing;Xu, Luang;Zhu, Yi;Guo, Tiannan
组织标本的高通量裂解和蛋白水解消化是临床蛋白质组学研究的主要瓶颈。在这里，我们描述了一个详细的方案压力循环技术（PCT）辅助样品制备的蛋白质组学分析活检组织。将重量约0.1-2 mg的新鲜冷冻或经福尔马林固定的石蜡包埋组织置于150 μL耐压管PCT-MicroTube中，并加入适当的裂解缓冲液。在用PCT-MicroPestle闭合后，将一批16个PCT-MicroTubes放置在Barocycler中，该Barocycler对样品施加振荡压力，从一个大气压到高达3000倍大气压。压力循环方案针对组织裂解和蛋白质消化进行了优化，并且可以在Barocycler中进行编程，从而为基于质谱的蛋白质组学提供可重复，稳健和高效的蛋白质提取和蛋白质水解消化。该方法不仅可以有效制备新鲜冷冻和福尔马林固定石蜡包埋组织，还可以制备细胞、粪便和泪液条。一次处理16个样品需要约3小时。所得肽可以通过各种基于质谱的蛋白质组学方法进行分析。我们演示了该方案在小鼠肾组织和八种人类肿瘤中的应用。组织标本的高通量裂解和蛋白水解消化是临床蛋白质组学研究的主要瓶颈。本方案描述了压力循环技术（PCT）辅助活检组织的样品制备。
High-throughput lysis and proteolytic digestion of biopsy-level tissue specimens is a major bottleneck for clinical proteomics. Here we describe a detailed protocol of pressure cycling technology (PCT)-assisted sample preparation for proteomic analysis of biopsy tissues. A piece of fresh frozen or formalin-fixed paraffin-embedded tissue weighing ~0.1–2 mg is placed in a 150 μL pressure-resistant tube called a PCT-MicroTube with proper lysis buffer. After closing with a PCT-MicroPestle, a batch of 16 PCT-MicroTubes are placed in a Barocycler, which imposes oscillating pressure to the samples from one atmosphere to up to ~3,000 times atmospheric pressure. The pressure cycling schemes are optimized for tissue lysis and protein digestion, and can be programmed in the Barocycler to allow reproducible, robust and efficient protein extraction and proteolysis digestion for mass spectrometry-based proteomics. This method allows effective preparation of not only fresh frozen and formalin-fixed paraffin-embedded tissue, but also cells, feces and tear strips. It takes ~3 h to process 16 samples in one batch. The resulting peptides can be analyzed by various mass spectrometry-based proteomics methods. We demonstrate the applications of this protocol with mouse kidney tissue and eight types of human tumors. High-throughput lysis and proteolytic digestion of biopsy-level tissue specimens is a major bottleneck for clinical proteomics. This protocol describes pressure cycling technology (PCT)-assisted sample preparation of biopsy tissues.
登录
查看更多内容
影响因子：
14.9
作者：
Ma J;Chen T;Wu S;Yang C;Bai M;Shu K;Li K;Zhang G;Jin Z;He F;Hermjakob H;Zhu Y
通讯作者：
Zhu Y
影响因子：
82.9
作者：
通讯作者：
--
影响因子：
7
作者：
Hood, BL;Darfler, MM;Krizman, DB
通讯作者：
Krizman, DB
影响因子：
7
作者：
Ross, PL;Huang, YLN;Pappin, DJ
通讯作者：
Pappin, DJ
影响因子：
7.4
作者：
Thompson, A;Schäfer, J;Hamon, C
通讯作者：
Hamon, C