Spatial Proteomics toward Subcellular Resolution by Coupling Deep Ultraviolet Laser Ablation with Nanodroplet Sample Preparation

IF 4.6 Q1 CHEMISTRY, ANALYTICAL ACS Measurement Science Au Pub Date : 2023-10-20 DOI:10.1021/acsmeasuresciau.3c00033
Piliang Xiang, Andrey Liyu, Yumi Kwon, Dehong Hu, Sarah M. Williams, Dušan Veličković, Lye Meng Markillie, William B. Chrisler, Ljiljana Paša-Tolić* and Ying Zhu*, 
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Abstract

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.

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将深紫外激光烧蚀与纳米液滴样品制备结合起来进行空间蛋白质组学研究,提高亚细胞分辨率
组织微环境的多重分子图谱分析(或称空间 omics)可提供有关细胞功能和疾病病理的重要见解。将激光显微切割与基于质谱的蛋白质组学相结合,可以对 1000 种蛋白质进行深入而无偏见的绘制。然而,激光显微切割的通量往往受到繁琐的两步程序、连续激光切割和样品采集的限制。两步程序还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。在此,我们开发了一种高通量、高分辨率的空间蛋白质组学平台,将深紫外(DUV)激光烧蚀(LA)与基于样品制备的纳米POTS(痕量样品的纳米液滴一次性处理)无缝结合。我们展示了 DUV-LA 系统能以每分钟 ∼30 个点的吞吐量和低至 2 μm 的空间分辨率从 10 μm 厚的人体胰腺组织切片中快速分离和采集组织样本。为了提高样品回收率,我们开发了一种近距离气溶胶收集方法,将 DMSO 液滴放置在靠近 LA 点的位置。我们证明,DUV-LA-nanoPOTS 平台能从直径分别为 7、13 和 19 μm 的烧蚀点平均检测到 1312、1533 和 1966 个蛋白质。在概念验证研究中,我们分离并分析了苏木精和伊红(H&E)染色显示的胰腺组织的两个不同亚细胞区域。定量蛋白质组学揭示了特异性富集于亚细胞区的蛋白质。
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来源期刊
ACS Measurement Science Au
ACS Measurement Science Au 化学计量学-
CiteScore
5.20
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0.00%
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0
期刊介绍: ACS Measurement Science Au is an open access journal that publishes experimental computational or theoretical research in all areas of chemical measurement science. Short letters comprehensive articles reviews and perspectives are welcome on topics that report on any phase of analytical operations including sampling measurement and data analysis. This includes:Chemical Reactions and SelectivityChemometrics and Data ProcessingElectrochemistryElemental and Molecular CharacterizationImagingInstrumentationMass SpectrometryMicroscale and Nanoscale systemsOmics (Genomics Proteomics Metabonomics Metabolomics and Bioinformatics)Sensors and Sensing (Biosensors Chemical Sensors Gas Sensors Intracellular Sensors Single-Molecule Sensors Cell Chips Arrays Microfluidic Devices)SeparationsSpectroscopySurface analysisPapers dealing with established methods need to offer a significantly improved original application of the method.
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