Homogeneous peptide-break assay for luminescent detection of enzymatic protein post-translational modification activity utilizing charged peptides
We have developed a rapid and sensitive universal peptide-based time-resolved luminescence assay for detection of enzymatic post-translational modifications (PTMs). PTMs play essential roles in intracellular signaling and cell regulation, thus providing functional protein diversity in cell. Due this...
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| Veröffentlicht in: | Analytica chimica acta 2019-05, Vol.1055, p.126-132 |
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| container_title | Analytica chimica acta |
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| creator | Kopra, Kari Tong-Ochoa, Natalia Laine, Mari Eskonen, Ville Koskinen, Päivi J. Härmä, Harri |
| description | We have developed a rapid and sensitive universal peptide-based time-resolved luminescence assay for detection of enzymatic post-translational modifications (PTMs). PTMs play essential roles in intracellular signaling and cell regulation, thus providing functional protein diversity in cell. Due this, impaired PTM patterns have been linked to multiple disease states. Clear link between PTMs and pathological conditions have also driven assay development further, but still today most of the methodologies are based on single-specificity or group-specific PTM-recognition. We have previously introduced leuzine-zipper based peptide-break technology as a viable option for universal PTM detection. Here, we introduce peptide-break technology utilizing single-label homogeneous quenching resonance energy transfer (QRET) and charge-based peptide-peptide interaction. We demonstrate the functionality of the new assay concept in phosphorylation, deacetylation, and citrullination. In a comparable study between previously introduced leucine-zipper and the novel charge-based approach, we found equal PTM detection performance and sensitivity, but the peptide design for new targets is simplified with the charged peptides. The new concept allows the use of short |
| doi_str_mv | 10.1016/j.aca.2018.12.041 |
| format | Article |
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•Antibody-free strategy for the detection of protein post-translational modifications (PTMs).•Charge-based peptides enable simple assay design and re-editing for different PTMs.•High throughput compatible method with nanomolar sensitivity for variety of PTMs.•Time-gated single-label luminescence monitoring enables low background and high S/B ratio.</description><identifier>ISSN: 0003-2670</identifier><identifier>EISSN: 1873-4324</identifier><identifier>DOI: 10.1016/j.aca.2018.12.041</identifier><identifier>PMID: 30782363</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>Acetylation ; Amino Acid Sequence ; Amino acids ; Assaying ; Charge transfer ; Citrullination ; Citrulline ; Coils ; Deacetylation ; Energy charge ; Energy transfer ; Europium - chemistry ; Intracellular signalling ; Leucine ; Leucine zipper proteins ; Luminescent Measurements - methods ; Peptide-break technology ; Peptides ; Peptides - chemistry ; Peptides - metabolism ; Phosphorylation ; Plates (structural members) ; Post-translation ; Protein post-translational modification ; Protein Processing, Post-Translational ; Proteins ; Technology ; Time-resolved luminescence ; Translation</subject><ispartof>Analytica chimica acta, 2019-05, Vol.1055, p.126-132</ispartof><rights>2018 Elsevier B.V.</rights><rights>Copyright © 2018 Elsevier B.V. All rights reserved.</rights><rights>Copyright Elsevier BV May 9, 2019</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c424t-4c6e0f1abe3547bd17da119cc6f2085c78053471f782bf622a855e03b62efdb53</citedby><cites>FETCH-LOGICAL-c424t-4c6e0f1abe3547bd17da119cc6f2085c78053471f782bf622a855e03b62efdb53</cites><orcidid>0000-0001-7585-6020</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0003267018314909$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3536,27903,27904,65309</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30782363$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kopra, Kari</creatorcontrib><creatorcontrib>Tong-Ochoa, Natalia</creatorcontrib><creatorcontrib>Laine, Mari</creatorcontrib><creatorcontrib>Eskonen, Ville</creatorcontrib><creatorcontrib>Koskinen, Päivi J.</creatorcontrib><creatorcontrib>Härmä, Harri</creatorcontrib><title>Homogeneous peptide-break assay for luminescent detection of enzymatic protein post-translational modification activity utilizing charged peptides</title><title>Analytica chimica acta</title><addtitle>Anal Chim Acta</addtitle><description>We have developed a rapid and sensitive universal peptide-based time-resolved luminescence assay for detection of enzymatic post-translational modifications (PTMs). PTMs play essential roles in intracellular signaling and cell regulation, thus providing functional protein diversity in cell. Due this, impaired PTM patterns have been linked to multiple disease states. Clear link between PTMs and pathological conditions have also driven assay development further, but still today most of the methodologies are based on single-specificity or group-specific PTM-recognition. We have previously introduced leuzine-zipper based peptide-break technology as a viable option for universal PTM detection. Here, we introduce peptide-break technology utilizing single-label homogeneous quenching resonance energy transfer (QRET) and charge-based peptide-peptide interaction. We demonstrate the functionality of the new assay concept in phosphorylation, deacetylation, and citrullination. In a comparable study between previously introduced leucine-zipper and the novel charge-based approach, we found equal PTM detection performance and sensitivity, but the peptide design for new targets is simplified with the charged peptides. The new concept allows the use of short <20 amino acid peptides without limitations rising from the leucine-zipper coiled-coil structure. Introduced methodology enables wash-free PTM detection in a 384-well plate format, using low nanomolar enzyme concentrations. Potentially, the peptide-break technique using charged peptides may be applicable for natural peptide sequences directly obtained from the target protein.
[Display omitted]
•Antibody-free strategy for the detection of protein post-translational modifications (PTMs).•Charge-based peptides enable simple assay design and re-editing for different PTMs.•High throughput compatible method with nanomolar sensitivity for variety of PTMs.•Time-gated single-label luminescence monitoring enables low background and high S/B ratio.</description><subject>Acetylation</subject><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Assaying</subject><subject>Charge transfer</subject><subject>Citrullination</subject><subject>Citrulline</subject><subject>Coils</subject><subject>Deacetylation</subject><subject>Energy charge</subject><subject>Energy transfer</subject><subject>Europium - chemistry</subject><subject>Intracellular signalling</subject><subject>Leucine</subject><subject>Leucine zipper proteins</subject><subject>Luminescent Measurements - methods</subject><subject>Peptide-break technology</subject><subject>Peptides</subject><subject>Peptides - chemistry</subject><subject>Peptides - metabolism</subject><subject>Phosphorylation</subject><subject>Plates (structural members)</subject><subject>Post-translation</subject><subject>Protein post-translational modification</subject><subject>Protein Processing, Post-Translational</subject><subject>Proteins</subject><subject>Technology</subject><subject>Time-resolved luminescence</subject><subject>Translation</subject><issn>0003-2670</issn><issn>1873-4324</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc1uFDEQhC0EIkvgAbggS1y4zOC2PT8RJxQFghQpl-Rseez24mVmPNieSJvH4InjzSYcOHCyWvqq2tVFyHtgNTBoP-9qbXTNGfQ18JpJeEE20HeikoLLl2TDGBMVbzt2Qt6ktCsjByZfkxPBup6LVmzIn8swhS3OGNZEF1yyt1gNEfUvqlPSe-pCpOM6-RmTwTlTixlN9mGmwVGc7_eTzt7QJYaMfqZLSLnKUc9p1AdKj3QK1jtvHkeqi_bO5z1dsx_9vZ-31PzUcYv2eXt6S145PSZ89_SekttvFzfnl9XV9fcf51-vKiO5zJU0LTIHekDRyG6w0FkNcGZM6zjrG9P1rBGyA1eiDq7lXPdNg0wMLUdnh0ackk9H3_L33yumrCZfMo6jfryG4tBLkOysgYJ-_AfdhTWWcIXiIEB2oukLBUfKxJBSRKeW6Ccd9wqYOhSmdqoUpg6FKeCqFFY0H56c12FC-1fx3FABvhwBLKe48xhVMh5ng9bHUoSywf_H_gHT2qma</recordid><startdate>20190509</startdate><enddate>20190509</enddate><creator>Kopra, Kari</creator><creator>Tong-Ochoa, Natalia</creator><creator>Laine, Mari</creator><creator>Eskonen, Ville</creator><creator>Koskinen, Päivi J.</creator><creator>Härmä, Harri</creator><general>Elsevier B.V</general><general>Elsevier BV</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>7QF</scope><scope>7QO</scope><scope>7QP</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7T7</scope><scope>7TA</scope><scope>7TB</scope><scope>7TK</scope><scope>7TM</scope><scope>7U5</scope><scope>7U7</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0001-7585-6020</orcidid></search><sort><creationdate>20190509</creationdate><title>Homogeneous peptide-break assay for luminescent detection of enzymatic protein post-translational modification activity utilizing charged peptides</title><author>Kopra, Kari ; Tong-Ochoa, Natalia ; Laine, Mari ; Eskonen, Ville ; Koskinen, Päivi J. ; Härmä, Harri</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c424t-4c6e0f1abe3547bd17da119cc6f2085c78053471f782bf622a855e03b62efdb53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Acetylation</topic><topic>Amino Acid Sequence</topic><topic>Amino acids</topic><topic>Assaying</topic><topic>Charge transfer</topic><topic>Citrullination</topic><topic>Citrulline</topic><topic>Coils</topic><topic>Deacetylation</topic><topic>Energy charge</topic><topic>Energy transfer</topic><topic>Europium - chemistry</topic><topic>Intracellular signalling</topic><topic>Leucine</topic><topic>Leucine zipper proteins</topic><topic>Luminescent Measurements - methods</topic><topic>Peptide-break technology</topic><topic>Peptides</topic><topic>Peptides - chemistry</topic><topic>Peptides - metabolism</topic><topic>Phosphorylation</topic><topic>Plates (structural members)</topic><topic>Post-translation</topic><topic>Protein post-translational modification</topic><topic>Protein Processing, Post-Translational</topic><topic>Proteins</topic><topic>Technology</topic><topic>Time-resolved luminescence</topic><topic>Translation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kopra, Kari</creatorcontrib><creatorcontrib>Tong-Ochoa, Natalia</creatorcontrib><creatorcontrib>Laine, Mari</creatorcontrib><creatorcontrib>Eskonen, Ville</creatorcontrib><creatorcontrib>Koskinen, Päivi J.</creatorcontrib><creatorcontrib>Härmä, Harri</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Aluminium Industry Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Electronics & Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Materials Business File</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Toxicology Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Analytica chimica acta</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kopra, Kari</au><au>Tong-Ochoa, Natalia</au><au>Laine, Mari</au><au>Eskonen, Ville</au><au>Koskinen, Päivi J.</au><au>Härmä, Harri</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Homogeneous peptide-break assay for luminescent detection of enzymatic protein post-translational modification activity utilizing charged peptides</atitle><jtitle>Analytica chimica acta</jtitle><addtitle>Anal Chim Acta</addtitle><date>2019-05-09</date><risdate>2019</risdate><volume>1055</volume><spage>126</spage><epage>132</epage><pages>126-132</pages><issn>0003-2670</issn><eissn>1873-4324</eissn><abstract>We have developed a rapid and sensitive universal peptide-based time-resolved luminescence assay for detection of enzymatic post-translational modifications (PTMs). PTMs play essential roles in intracellular signaling and cell regulation, thus providing functional protein diversity in cell. Due this, impaired PTM patterns have been linked to multiple disease states. Clear link between PTMs and pathological conditions have also driven assay development further, but still today most of the methodologies are based on single-specificity or group-specific PTM-recognition. We have previously introduced leuzine-zipper based peptide-break technology as a viable option for universal PTM detection. Here, we introduce peptide-break technology utilizing single-label homogeneous quenching resonance energy transfer (QRET) and charge-based peptide-peptide interaction. We demonstrate the functionality of the new assay concept in phosphorylation, deacetylation, and citrullination. In a comparable study between previously introduced leucine-zipper and the novel charge-based approach, we found equal PTM detection performance and sensitivity, but the peptide design for new targets is simplified with the charged peptides. The new concept allows the use of short <20 amino acid peptides without limitations rising from the leucine-zipper coiled-coil structure. Introduced methodology enables wash-free PTM detection in a 384-well plate format, using low nanomolar enzyme concentrations. Potentially, the peptide-break technique using charged peptides may be applicable for natural peptide sequences directly obtained from the target protein.
[Display omitted]
•Antibody-free strategy for the detection of protein post-translational modifications (PTMs).•Charge-based peptides enable simple assay design and re-editing for different PTMs.•High throughput compatible method with nanomolar sensitivity for variety of PTMs.•Time-gated single-label luminescence monitoring enables low background and high S/B ratio.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>30782363</pmid><doi>10.1016/j.aca.2018.12.041</doi><tpages>7</tpages><orcidid>https://orcid.org/0000-0001-7585-6020</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Acetylation Amino Acid Sequence Amino acids Assaying Charge transfer Citrullination Citrulline Coils Deacetylation Energy charge Energy transfer Europium - chemistry Intracellular signalling Leucine Leucine zipper proteins Luminescent Measurements - methods Peptide-break technology Peptides Peptides - chemistry Peptides - metabolism Phosphorylation Plates (structural members) Post-translation Protein post-translational modification Protein Processing, Post-Translational Proteins Technology Time-resolved luminescence Translation |
| title | Homogeneous peptide-break assay for luminescent detection of enzymatic protein post-translational modification activity utilizing charged peptides |
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