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使用邻苯二醛交联剂的快速交联质谱法对蛋白质展开的特性分析。

Characterization of protein unfolding by fast cross-linking mass spectrometry using di-ortho-phthalaldehyde cross-linkers.

机构信息

National Institute of Biological Sciences (NIBS), 102206, Beijing, China.

Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, 102206, Beijing, China.

出版信息

Nat Commun. 2022 Mar 18;13(1):1468. doi: 10.1038/s41467-022-28879-4.

DOI:10.1038/s41467-022-28879-4
PMID:35304446
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8933431/
Abstract

Chemical cross-linking of proteins coupled with mass spectrometry is widely used in protein structural analysis. In this study we develop a class of non-hydrolyzable amine-selective di-ortho-phthalaldehyde (DOPA) cross-linkers, one of which is called DOPA2. Cross-linking of proteins with DOPA2 is 60-120 times faster than that with the N-hydroxysuccinimide ester cross-linker DSS. Compared with DSS cross-links, DOPA2 cross-links show better agreement with the crystal structures of tested proteins. More importantly, DOPA2 has unique advantages when working at low pH, low temperature, or in the presence of denaturants. Using staphylococcal nuclease, bovine serum albumin, and bovine pancreatic ribonuclease A, we demonstrate that DOPA2 cross-linking provides abundant spatial information about the conformations of progressively denatured forms of these proteins. Furthermore, DOPA2 cross-linking allows time-course analysis of protein conformational changes during denaturant-induced unfolding.

摘要

蛋白质的化学交联与质谱联用广泛应用于蛋白质结构分析。本研究开发了一类非水解性伯胺选择性二邻苯二甲醛（DOPA）交联剂，其中一种称为 DOPA2。DOPA2 与蛋白质的交联速度比 N-羟基琥珀酰亚胺酯交联剂 DSS 快 60-120 倍。与 DSS 交联相比，DOPA2 交联与测试蛋白的晶体结构更吻合。更重要的是，DOPA2 在低 pH 值、低温或变性剂存在下具有独特的优势。使用金黄色葡萄球菌核酸酶、牛血清白蛋白和牛胰腺核糖核酸酶 A，我们证明 DOPA2 交联提供了关于这些蛋白质逐渐变性形式构象的丰富空间信息。此外，DOPA2 交联允许在变性剂诱导解折叠过程中进行蛋白质构象变化的时程分析。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/4007eb5d9cd7/41467_2022_28879_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/c64cbd68dbdb/41467_2022_28879_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/08d70669354a/41467_2022_28879_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/6f1113e6689d/41467_2022_28879_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/6c049718eedd/41467_2022_28879_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/17026dbe91f8/41467_2022_28879_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/f072653ada4c/41467_2022_28879_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/d6bc36e5c3f5/41467_2022_28879_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/4007eb5d9cd7/41467_2022_28879_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/c64cbd68dbdb/41467_2022_28879_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/08d70669354a/41467_2022_28879_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/6f1113e6689d/41467_2022_28879_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/6c049718eedd/41467_2022_28879_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/17026dbe91f8/41467_2022_28879_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/f072653ada4c/41467_2022_28879_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/d6bc36e5c3f5/41467_2022_28879_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b794/8933431/4007eb5d9cd7/41467_2022_28879_Fig8_HTML.jpg

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