A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues
Hydrogen peroxide (H 2 O 2 ) is a key member of the reactive oxygen species family of transient small molecules that has broad contributions to oxidative stress and redox signaling. The development of selective and sensitive chemical probes can enable the study of H 2 O 2 biology in cell, tissue and...
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| Veröffentlicht in: | Nature protocols 2022-07, Vol.17 (7), p.1691-1710 |
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| creator | Hoshi, Kaede Messina, Marco S. Ohata, Jun Chung, Clive Yik-Sham Chang, Christopher J. |
| description | Hydrogen peroxide (H
2
O
2
) is a key member of the reactive oxygen species family of transient small molecules that has broad contributions to oxidative stress and redox signaling. The development of selective and sensitive chemical probes can enable the study of H
2
O
2
biology in cell, tissue and animal models. Peroxymycin-1 is a histochemical activity–based sensing probe that responds to H
2
O
2
via chemoselective boronate oxidation to release puromycin, which is then covalently incorporated into nascent proteins by the ribosome and can be detected by antibody staining. Here, we describe an optimized two-step, one-pot protocol for synthesizing Peroxymycin-1 with improved yields over our originally reported procedure. We also present detailed procedures for applying Peroxymycin-1 to a broad range of biological samples spanning cells to animal tissues for profiling H
2
O
2
levels through histochemical detection by using commercially available anti-puromycin antibodies. The preparation of Peroxymycin-1 takes 9 h, the confocal imaging experiments of endogenous H
2
O
2
levels across different cancer cell lines take 1 d, the dot blot analysis of mouse liver tissues takes 1 d and the confocal imaging of mouse liver tissues takes 3–4 d.
This protocol details the synthesis and use of Peroxymycin-1, an activity-based histochemical probe for hydrogen peroxide detection in fixed cell and mouse tissue samples. |
| doi_str_mv | 10.1038/s41596-022-00694-7 |
| format | Article |
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2
O
2
) is a key member of the reactive oxygen species family of transient small molecules that has broad contributions to oxidative stress and redox signaling. The development of selective and sensitive chemical probes can enable the study of H
2
O
2
biology in cell, tissue and animal models. Peroxymycin-1 is a histochemical activity–based sensing probe that responds to H
2
O
2
via chemoselective boronate oxidation to release puromycin, which is then covalently incorporated into nascent proteins by the ribosome and can be detected by antibody staining. Here, we describe an optimized two-step, one-pot protocol for synthesizing Peroxymycin-1 with improved yields over our originally reported procedure. We also present detailed procedures for applying Peroxymycin-1 to a broad range of biological samples spanning cells to animal tissues for profiling H
2
O
2
levels through histochemical detection by using commercially available anti-puromycin antibodies. The preparation of Peroxymycin-1 takes 9 h, the confocal imaging experiments of endogenous H
2
O
2
levels across different cancer cell lines take 1 d, the dot blot analysis of mouse liver tissues takes 1 d and the confocal imaging of mouse liver tissues takes 3–4 d.
This protocol details the synthesis and use of Peroxymycin-1, an activity-based histochemical probe for hydrogen peroxide detection in fixed cell and mouse tissue samples.</description><identifier>ISSN: 1754-2189</identifier><identifier>EISSN: 1750-2799</identifier><identifier>DOI: 10.1038/s41596-022-00694-7</identifier><identifier>PMID: 35562423</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>631/1647/1888 ; 631/92 ; 639/638/92 ; Analytical Chemistry ; Animal models ; Animal tissues ; Animals ; Antibodies ; Biological properties ; Biological samples ; Biological Techniques ; Biomedical and Life Sciences ; Cell culture ; Computational Biology/Bioinformatics ; Fluorescent Dyes - chemistry ; Hydrogen peroxide ; Hydrogen Peroxide - analysis ; Life Sciences ; Liver ; Mice ; Microarrays ; Organic Chemistry ; Oxidation ; Oxidation-Reduction ; Oxidative stress ; Protocol ; Puromycin ; Reactive oxygen species ; Signal Transduction ; Staining ; Staining and Labeling ; Tumor cell lines</subject><ispartof>Nature protocols, 2022-07, Vol.17 (7), p.1691-1710</ispartof><rights>Springer Nature Limited 2022</rights><rights>2022. Springer Nature Limited.</rights><rights>Springer Nature Limited 2022.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c475t-f239e3dddca0a2729c5e194f29f3e5ddd95df56df95690a4562f130fcc4ada73</citedby><cites>FETCH-LOGICAL-c475t-f239e3dddca0a2729c5e194f29f3e5ddd95df56df95690a4562f130fcc4ada73</cites><orcidid>0000-0003-1382-719X ; 0000-0002-3614-7472 ; 0000-0001-5732-9497 ; 0000-0003-2827-118X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/s41596-022-00694-7$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/s41596-022-00694-7$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,314,776,780,881,27901,27902,41464,42533,51294</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/35562423$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hoshi, Kaede</creatorcontrib><creatorcontrib>Messina, Marco S.</creatorcontrib><creatorcontrib>Ohata, Jun</creatorcontrib><creatorcontrib>Chung, Clive Yik-Sham</creatorcontrib><creatorcontrib>Chang, Christopher J.</creatorcontrib><title>A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues</title><title>Nature protocols</title><addtitle>Nat Protoc</addtitle><addtitle>Nat Protoc</addtitle><description>Hydrogen peroxide (H
2
O
2
) is a key member of the reactive oxygen species family of transient small molecules that has broad contributions to oxidative stress and redox signaling. The development of selective and sensitive chemical probes can enable the study of H
2
O
2
biology in cell, tissue and animal models. Peroxymycin-1 is a histochemical activity–based sensing probe that responds to H
2
O
2
via chemoselective boronate oxidation to release puromycin, which is then covalently incorporated into nascent proteins by the ribosome and can be detected by antibody staining. Here, we describe an optimized two-step, one-pot protocol for synthesizing Peroxymycin-1 with improved yields over our originally reported procedure. We also present detailed procedures for applying Peroxymycin-1 to a broad range of biological samples spanning cells to animal tissues for profiling H
2
O
2
levels through histochemical detection by using commercially available anti-puromycin antibodies. The preparation of Peroxymycin-1 takes 9 h, the confocal imaging experiments of endogenous H
2
O
2
levels across different cancer cell lines take 1 d, the dot blot analysis of mouse liver tissues takes 1 d and the confocal imaging of mouse liver tissues takes 3–4 d.
This protocol details the synthesis and use of Peroxymycin-1, an activity-based histochemical probe for hydrogen peroxide detection in fixed cell and mouse tissue samples.</description><subject>631/1647/1888</subject><subject>631/92</subject><subject>639/638/92</subject><subject>Analytical Chemistry</subject><subject>Animal models</subject><subject>Animal tissues</subject><subject>Animals</subject><subject>Antibodies</subject><subject>Biological properties</subject><subject>Biological samples</subject><subject>Biological Techniques</subject><subject>Biomedical and Life Sciences</subject><subject>Cell culture</subject><subject>Computational Biology/Bioinformatics</subject><subject>Fluorescent Dyes - chemistry</subject><subject>Hydrogen peroxide</subject><subject>Hydrogen Peroxide - analysis</subject><subject>Life Sciences</subject><subject>Liver</subject><subject>Mice</subject><subject>Microarrays</subject><subject>Organic Chemistry</subject><subject>Oxidation</subject><subject>Oxidation-Reduction</subject><subject>Oxidative stress</subject><subject>Protocol</subject><subject>Puromycin</subject><subject>Reactive oxygen species</subject><subject>Signal Transduction</subject><subject>Staining</subject><subject>Staining and Labeling</subject><subject>Tumor cell lines</subject><issn>1754-2189</issn><issn>1750-2799</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp9kctuHCEQRVEUy-8fyCJCyhqHZ3ezsiwrsSNZ8sZ7xEAxg9UDHeixPJt8exiP7SSbLBCIOnXrli5Cnxi9YFQMX6tkSneEck4o7bQk_Qd0zHpFCe-1_vjyloSzQR-hk1ofKZW96PpDdCSU6rjk4hj9usLTpuT11sVEPEyQPKQZWzfHpzhvycJW8LhCqjEt8VTyAnDIBa9inbNbwTo6O-I625h2QA54tfUlLyHhCUp-jh5wTNjBOFZsk28nrlvHHGvdQD1DB8GOFc5f71P08P3bw_Utubu_-XF9dUec7NVMAhcahPfeWWp5z7VTwLQMXAcBqv1r5YPqfNCq09TKtl1gggbnpPW2F6foci87bRZr8K6tWOxoptK8lK3JNpp_KymuzDI_GUa5EKJjTeHLq0LJP5vx2TzmTUnNs-HdoAbBxMAbxfeUK7nWAuF9BKNml5nZZ2ZaZuYlM7Mz9_lvc-8tbyE1QOyB2kppCeXP7P_I_gbpb6a3</recordid><startdate>20220701</startdate><enddate>20220701</enddate><creator>Hoshi, Kaede</creator><creator>Messina, Marco S.</creator><creator>Ohata, Jun</creator><creator>Chung, Clive Yik-Sham</creator><creator>Chang, Christopher J.</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QG</scope><scope>7T5</scope><scope>7T7</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PATMY</scope><scope>PHGZM</scope><scope>PHGZT</scope><scope>PJZUB</scope><scope>PKEHL</scope><scope>PPXIY</scope><scope>PQEST</scope><scope>PQGLB</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0003-1382-719X</orcidid><orcidid>https://orcid.org/0000-0002-3614-7472</orcidid><orcidid>https://orcid.org/0000-0001-5732-9497</orcidid><orcidid>https://orcid.org/0000-0003-2827-118X</orcidid></search><sort><creationdate>20220701</creationdate><title>A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues</title><author>Hoshi, Kaede ; Messina, Marco S. ; Ohata, Jun ; Chung, Clive Yik-Sham ; Chang, Christopher J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c475t-f239e3dddca0a2729c5e194f29f3e5ddd95df56df95690a4562f130fcc4ada73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>631/1647/1888</topic><topic>631/92</topic><topic>639/638/92</topic><topic>Analytical Chemistry</topic><topic>Animal models</topic><topic>Animal tissues</topic><topic>Animals</topic><topic>Antibodies</topic><topic>Biological properties</topic><topic>Biological samples</topic><topic>Biological Techniques</topic><topic>Biomedical and Life Sciences</topic><topic>Cell culture</topic><topic>Computational Biology/Bioinformatics</topic><topic>Fluorescent Dyes - chemistry</topic><topic>Hydrogen peroxide</topic><topic>Hydrogen Peroxide - analysis</topic><topic>Life Sciences</topic><topic>Liver</topic><topic>Mice</topic><topic>Microarrays</topic><topic>Organic Chemistry</topic><topic>Oxidation</topic><topic>Oxidation-Reduction</topic><topic>Oxidative stress</topic><topic>Protocol</topic><topic>Puromycin</topic><topic>Reactive oxygen species</topic><topic>Signal Transduction</topic><topic>Staining</topic><topic>Staining and Labeling</topic><topic>Tumor cell lines</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hoshi, Kaede</creatorcontrib><creatorcontrib>Messina, Marco S.</creatorcontrib><creatorcontrib>Ohata, Jun</creatorcontrib><creatorcontrib>Chung, Clive Yik-Sham</creatorcontrib><creatorcontrib>Chang, Christopher J.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Immunology Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>ProQuest Central (New)</collection><collection>ProQuest One Academic (New)</collection><collection>ProQuest Health & Medical Research Collection</collection><collection>ProQuest One Academic Middle East (New)</collection><collection>ProQuest One Health & Nursing</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Applied & Life Sciences</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nature protocols</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hoshi, Kaede</au><au>Messina, Marco S.</au><au>Ohata, Jun</au><au>Chung, Clive Yik-Sham</au><au>Chang, Christopher J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues</atitle><jtitle>Nature protocols</jtitle><stitle>Nat Protoc</stitle><addtitle>Nat Protoc</addtitle><date>2022-07-01</date><risdate>2022</risdate><volume>17</volume><issue>7</issue><spage>1691</spage><epage>1710</epage><pages>1691-1710</pages><issn>1754-2189</issn><eissn>1750-2799</eissn><abstract>Hydrogen peroxide (H
2
O
2
) is a key member of the reactive oxygen species family of transient small molecules that has broad contributions to oxidative stress and redox signaling. The development of selective and sensitive chemical probes can enable the study of H
2
O
2
biology in cell, tissue and animal models. Peroxymycin-1 is a histochemical activity–based sensing probe that responds to H
2
O
2
via chemoselective boronate oxidation to release puromycin, which is then covalently incorporated into nascent proteins by the ribosome and can be detected by antibody staining. Here, we describe an optimized two-step, one-pot protocol for synthesizing Peroxymycin-1 with improved yields over our originally reported procedure. We also present detailed procedures for applying Peroxymycin-1 to a broad range of biological samples spanning cells to animal tissues for profiling H
2
O
2
levels through histochemical detection by using commercially available anti-puromycin antibodies. The preparation of Peroxymycin-1 takes 9 h, the confocal imaging experiments of endogenous H
2
O
2
levels across different cancer cell lines take 1 d, the dot blot analysis of mouse liver tissues takes 1 d and the confocal imaging of mouse liver tissues takes 3–4 d.
This protocol details the synthesis and use of Peroxymycin-1, an activity-based histochemical probe for hydrogen peroxide detection in fixed cell and mouse tissue samples.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>35562423</pmid><doi>10.1038/s41596-022-00694-7</doi><tpages>20</tpages><orcidid>https://orcid.org/0000-0003-1382-719X</orcidid><orcidid>https://orcid.org/0000-0002-3614-7472</orcidid><orcidid>https://orcid.org/0000-0001-5732-9497</orcidid><orcidid>https://orcid.org/0000-0003-2827-118X</orcidid><oa>free_for_read</oa></addata></record> |
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| ispartof | Nature protocols, 2022-07, Vol.17 (7), p.1691-1710 |
| issn | 1754-2189 1750-2799 |
| language | eng |
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| source | MEDLINE; Springer Nature - Complete Springer Journals; Nature |
| subjects | 631/1647/1888 631/92 639/638/92 Analytical Chemistry Animal models Animal tissues Animals Antibodies Biological properties Biological samples Biological Techniques Biomedical and Life Sciences Cell culture Computational Biology/Bioinformatics Fluorescent Dyes - chemistry Hydrogen peroxide Hydrogen Peroxide - analysis Life Sciences Liver Mice Microarrays Organic Chemistry Oxidation Oxidation-Reduction Oxidative stress Protocol Puromycin Reactive oxygen species Signal Transduction Staining Staining and Labeling Tumor cell lines |
| title | A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues |
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