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时门控拉曼光谱法和缺氧与常氧肾癌细胞外囊泡的蛋白质组学分析。

Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles.

机构信息

Laboratory of Developmental Biology, Disease Networks Research Unit, Faculty of Biochemistry and Molecular Medicine, University of Oulu and Kvantum Institute, 90014, Oulu, Finland.

VTT Technical Research Centre of Finland, 90570, Oulu, Finland.

出版信息

Sci Rep. 2021 Oct 1;11(1):19594. doi: 10.1038/s41598-021-99004-6.

DOI:10.1038/s41598-021-99004-6
PMID:34599227
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8486794/
Abstract

Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell-cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols.

摘要

细胞外囊泡 (EVs) 是一类参与细胞间通讯的具有不同功能的小膜包裹颗粒，但用于鉴定 EVs 的技术仍然有限。缺氧是实体瘤中的一种典型情况，而源自肿瘤的 EVs 通过肿瘤细胞支持肿瘤生长和组织侵袭。我们发现，与常氧相比，肾腺癌细胞暴露于缺氧会诱导 EV 分泌，并导致 EV 蛋白货物发生明显变化。蛋白质组学分析表明，缺氧 EV 样本中参与黏附的蛋白质，如整合素，过表达。我们进一步评估了时间门控拉曼光谱 (TG-RS) 和表面增强时间门控拉曼光谱 (TG-SERS) 来表征 EVs。虽然传统的连续波激发 Raman 光谱没有提供明显的信号，但 TG-RS 获得了显著的信号，并且在 TG-SERS 中进一步增强。Raman 信号由于 EV 蛋白的化学键发生变化，在酰胺区域显示出特征性的变化。结果表明，TG-RS 和 TG-SERS 是很有前途的无标记技术，可以研究外部刺激（如缺氧）对 EV 产生的细胞影响，以及源自不同 EV 纯化方案的差异。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/17b0/8486794/22a745718efa/41598_2021_99004_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/17b0/8486794/0300841c0324/41598_2021_99004_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/17b0/8486794/308d04d886d9/41598_2021_99004_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/17b0/8486794/a0e4834e921d/41598_2021_99004_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/17b0/8486794/c1ceb62e4d07/41598_2021_99004_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/17b0/8486794/22a745718efa/41598_2021_99004_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/17b0/8486794/0300841c0324/41598_2021_99004_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/17b0/8486794/308d04d886d9/41598_2021_99004_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/17b0/8486794/a0e4834e921d/41598_2021_99004_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/17b0/8486794/c1ceb62e4d07/41598_2021_99004_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/17b0/8486794/22a745718efa/41598_2021_99004_Fig5_HTML.jpg

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