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基于点击化学的方法检测原代小鼠巨噬细胞中4-辛基衣康酸酯烷基化蛋白

Click-chemistry-based protocol for detecting 4-octyl-itaconate-alkylated proteins in primary mouse macrophages.

作者信息

Su Chaofei, Cheng Tian, Zhang Hanyi, Yin Hang

机构信息

State Key laboratory of Membrane Biology, School of Pharmaceutical Sciences, Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, China.

State Key laboratory of Membrane Biology, School of Pharmaceutical Sciences, Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, China.

出版信息

STAR Protoc. 2024 Dec 20;5(4):103314. doi: 10.1016/j.xpro.2024.103314. Epub 2024 Sep 19.

DOI:10.1016/j.xpro.2024.103314
PMID:39298323
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11426128/
Abstract

4-Octyl itaconate (4-OI), a derivative of itaconate, inhibits inflammation by alkylating its target proteins. Here, we present a click-chemistry-based protocol for detecting 4-OI-alkylated proteins in mouse primary bone-marrow-derived macrophages (BMDMs) by using an itaconate-alkyne (ITalk) probe. We describe steps for culturing and treating BMDMs and details on using click chemistry in the cell lysate. We also detail procedures for detecting alkylated proteins by western blot. For complete details on the use and execution of this protocol, please refer to Su et al..

摘要

衣康酸4-辛酯（4-OI）是衣康酸的衍生物，通过烷基化其靶蛋白来抑制炎症。在此，我们提出了一种基于点击化学的方案，用于通过使用衣康酸炔烃（ITalk）探针在小鼠原代骨髓来源巨噬细胞（BMDM）中检测4-OI烷基化蛋白。我们描述了培养和处理BMDM的步骤以及在细胞裂解物中使用点击化学的细节。我们还详细说明了通过蛋白质印迹检测烷基化蛋白的程序。有关本方案使用和执行的完整详细信息，请参考Su等人的研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e2e6/11426128/c38223111367/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e2e6/11426128/bf814c9a2f9f/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e2e6/11426128/77176f7132b0/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e2e6/11426128/c38223111367/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e2e6/11426128/bf814c9a2f9f/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e2e6/11426128/77176f7132b0/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e2e6/11426128/c38223111367/gr2.jpg

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1
Click-chemistry-based protocol for detecting 4-octyl-itaconate-alkylated proteins in primary mouse macrophages.基于点击化学的方法检测原代小鼠巨噬细胞中4-辛基衣康酸酯烷基化蛋白
STAR Protoc. 2024 Dec 20;5(4):103314. doi: 10.1016/j.xpro.2024.103314. Epub 2024 Sep 19.
2
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本文引用的文献

1
Itaconate in host inflammation and defense.衣康酸在宿主炎症和防御中的作用。
Trends Endocrinol Metab. 2024 Jul;35(7):586-606. doi: 10.1016/j.tem.2024.02.004. Epub 2024 Mar 5.
2
4-Octyl itaconate restricts STING activation by blocking its palmitoylation.4-辛烯酸抑制 STING 的棕榈酰化来限制其激活。
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Metabolic labeling and LC-MS/MS-based identification of interleukin-1α-induced secreted proteomes from epithelial cells in the presence or absence of serum.
在有或无血清存在的情况下，通过代谢标记和基于液相色谱-串联质谱法鉴定白细胞介素-1α诱导上皮细胞分泌的蛋白质组。
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Click-chemistry-based protocol to quantitatively assess fatty acid uptake by Mycobacterium tuberculosis in axenic culture and inside mouse macrophages.基于点击化学的方案定量评估分枝杆菌在体外培养和小鼠巨噬细胞内的脂肪酸摄取。
STAR Protoc. 2023 Mar 17;4(1):102062. doi: 10.1016/j.xpro.2023.102062. Epub 2023 Jan 24.
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A metabolic labeling protocol to enrich myristoylated proteins from .一种代谢标记方法，用于从. 中富集豆蔻酰化蛋白。
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Chemoproteomic profiling of itaconations in .衣康酸的化学蛋白质组学分析
Chem Sci. 2021 Mar 31;12(17):6059-6063. doi: 10.1039/d1sc00660f.
7
Preparation and culture of bone marrow-derived macrophages from mice for functional analysis.从小鼠中制备和培养骨髓来源的巨噬细胞用于功能分析。
STAR Protoc. 2020 Dec 31;2(1):100246. doi: 10.1016/j.xpro.2020.100246. eCollection 2021 Mar 19.
8
Succination inactivates gasdermin D and blocks pyroptosis.琥珀酰化使gasdermin D失活并阻断细胞焦亡。
Science. 2020 Sep 25;369(6511):1633-1637. doi: 10.1126/science.abb9818. Epub 2020 Aug 20.
9
Chemoproteomic Profiling of Itaconation by Bioorthogonal Probes in Inflammatory Macrophages.通过生物正交探针在炎症巨噬细胞中对衣康酸的化学蛋白质组学分析。
J Am Chem Soc. 2020 Jun 24;142(25):10894-10898. doi: 10.1021/jacs.9b11962. Epub 2020 Jun 11.
10
Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.衣康酸是一种抗炎代谢物，通过对 KEAP1 的烷基化作用激活 Nrf2。
Nature. 2018 Apr 5;556(7699):113-117. doi: 10.1038/nature25986. Epub 2018 Mar 28.