Spatial Proteomics toward Subcellular Resolution by Coupling Deep Ultraviolet Laser Ablation with Nanodroplet Sample Preparation.
Spatial Proteomics toward Subcellular Resolution by Coupling Deep Ultraviolet Laser Ablation with Nanodroplet Sample Preparation.
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DOI：
10.1021/acsmeasuresciau.3c00033
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发表时间：
2023-12-20
期刊：
影响因子：
--
通讯作者：
Zhu, Ying
中科院分区：
文献类型：
--
作者：
Xiang, Piliang;Liyu, Andrey;Kwon, Yumi;Hu, Dehong;Williams, Sarah M;Velickovic, Dusan;Markillie, Lye Meng;Chrisler, William B;Pasa-Tolic, Ljiljana;Zhu, Ying
组织微环境的多重分子分析或空间组学可以为细胞功能和疾病病理学提供重要见解。激光显微切割与基于质谱的蛋白质组学的结合已经能够对>1000种蛋白质进行深入和无偏见的映射。然而，由于繁琐的两步程序，连续的激光切割和样品收集，激光显微切割的吞吐量往往是有限的。这两步程序也阻碍了亚细胞蛋白质组学所需的空间分辨率进一步提高到<10 μm。在这里，我们开发了一个高通量和高分辨率的空间蛋白质组学平台，通过无缝耦合深紫外（DUV）激光消融（LA）与纳米POTS（纳米液滴处理在一锅微量样品）为基础的样品制备。我们证明了DUV-LA系统可以快速分离和收集组织样品，其通量为10 μ m/min，空间分辨率低至2 μm，来自10 μm厚的人胰腺组织切片。为了提高样品回收率，我们通过将DMSO液滴放置在LA点附近开发了一种邻近气溶胶收集方法。我们证明了DUV-LA-nanoPOTS平台可以分别从直径为7、13和19 μm的消融点中检测平均1312、1533和1966个蛋白质。在一项概念验证研究中，我们分离并分析了通过苏木精和伊红（H&E）染色显示的胰腺组织的两个不同的亚细胞区域。定量蛋白质组学揭示了蛋白质特异性富集到亚细胞区室。
Multiplexed molecular profiling of tissue microenvironments, or spatial omics, can provide critical insights into cellular functions and disease pathology. The coupling of laser microdissection with mass spectrometry-based proteomics has enabled deep and unbiased mapping of >1000 proteins. However, the throughput of laser microdissection is often limited due to tedious two-step procedures, sequential laser cutting, and sample collection. The two-step procedure also hinders the further improvement of spatial resolution to <10 μm as needed for subcellular proteomics. Herein, we developed a high-throughput and high-resolution spatial proteomics platform by seamlessly coupling deep ultraviolet (DUV) laser ablation (LA) with nanoPOTS (Nanodroplet Processing in One pot for Trace Samples)-based sample preparation. We demonstrated the DUV-LA system can quickly isolate and collect tissue samples at a throughput of ∼30 spots/min and a spatial resolution down to 2 μm from a 10 μm thick human pancreas tissue section. To improve sample recovery, we developed a proximity aerosol collection approach by placing DMSO droplets close to LA spots. We demonstrated the DUV-LA-nanoPOTS platform can detect an average of 1312, 1533, and 1966 proteins from ablation spots with diameters of 7, 13, and 19 μm, respectively. In a proof-of-concept study, we isolated and profiled two distinct subcellular regions of the pancreas tissue revealed by hematoxylin and eosin (H&E) staining. Quantitative proteomics revealed proteins specifically enriched to subcellular compartments.
影响因子：
7.4
作者：
Frost DC;Rust CJ;Robinson RAS;Li L
通讯作者：
Li L