Engineered ChymotrypsiN for Mass Spectrometry-Based Detection of Protein Glycosylation
We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an...
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| Veröffentlicht in: | ACS chemical biology 2019-12, Vol.14 (12), p.2616-2628 |
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| container_title | ACS chemical biology |
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| creator | Ramesh, Balakrishnan Abnouf, Shaza Mali, Sujina Moree, Wilna J Patil, Ujwal Bark, Steven J Varadarajan, Navin |
| description | We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn-containing substrates. We confirmed that protein engineering did not compromise the stability of the enzyme by biophysical characterization. Comparison of wild-type ChyB and ChyB-Asn in profiling lysates of HEK293 cells demonstrated both qualitative and quantitative differences in the nature of the peptides and proteins identified by liquid chromatography and tandem mass spectrometry. ChyB-Asn enabled the identification of partially glycosylated Asn sites within a model glycoprotein and in the extracellular proteome of Jurkat T cells. ChymotrypsiN is a valuable addition to the toolkit of proteases to aid the mapping of N-linked glycosylation sites within proteins and proteomes. |
| doi_str_mv | 10.1021/acschembio.9b00506 |
| format | Article |
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The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn-containing substrates. We confirmed that protein engineering did not compromise the stability of the enzyme by biophysical characterization. Comparison of wild-type ChyB and ChyB-Asn in profiling lysates of HEK293 cells demonstrated both qualitative and quantitative differences in the nature of the peptides and proteins identified by liquid chromatography and tandem mass spectrometry. ChyB-Asn enabled the identification of partially glycosylated Asn sites within a model glycoprotein and in the extracellular proteome of Jurkat T cells. ChymotrypsiN is a valuable addition to the toolkit of proteases to aid the mapping of N-linked glycosylation sites within proteins and proteomes.</description><identifier>ISSN: 1554-8929</identifier><identifier>EISSN: 1554-8937</identifier><identifier>DOI: 10.1021/acschembio.9b00506</identifier><identifier>PMID: 31710461</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Chymotrypsin - genetics ; Chymotrypsin - metabolism ; Escherichia coli - genetics ; Glycosylation ; High-Throughput Screening Assays ; Humans ; Mass Spectrometry - methods ; Substrate Specificity</subject><ispartof>ACS chemical biology, 2019-12, Vol.14 (12), p.2616-2628</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a342t-868b6189b46afdc9779ca69af8554b736822163dff7665cb898bc1f24a070e6e3</citedby><cites>FETCH-LOGICAL-a342t-868b6189b46afdc9779ca69af8554b736822163dff7665cb898bc1f24a070e6e3</cites><orcidid>0000-0001-7524-8228</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/acschembio.9b00506$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/acschembio.9b00506$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>315,781,785,2766,27080,27928,27929,56742,56792</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31710461$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ramesh, Balakrishnan</creatorcontrib><creatorcontrib>Abnouf, Shaza</creatorcontrib><creatorcontrib>Mali, Sujina</creatorcontrib><creatorcontrib>Moree, Wilna J</creatorcontrib><creatorcontrib>Patil, Ujwal</creatorcontrib><creatorcontrib>Bark, Steven J</creatorcontrib><creatorcontrib>Varadarajan, Navin</creatorcontrib><title>Engineered ChymotrypsiN for Mass Spectrometry-Based Detection of Protein Glycosylation</title><title>ACS chemical biology</title><addtitle>ACS Chem. Biol</addtitle><description>We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn-containing substrates. We confirmed that protein engineering did not compromise the stability of the enzyme by biophysical characterization. Comparison of wild-type ChyB and ChyB-Asn in profiling lysates of HEK293 cells demonstrated both qualitative and quantitative differences in the nature of the peptides and proteins identified by liquid chromatography and tandem mass spectrometry. ChyB-Asn enabled the identification of partially glycosylated Asn sites within a model glycoprotein and in the extracellular proteome of Jurkat T cells. ChymotrypsiN is a valuable addition to the toolkit of proteases to aid the mapping of N-linked glycosylation sites within proteins and proteomes.</description><subject>Chymotrypsin - genetics</subject><subject>Chymotrypsin - metabolism</subject><subject>Escherichia coli - genetics</subject><subject>Glycosylation</subject><subject>High-Throughput Screening Assays</subject><subject>Humans</subject><subject>Mass Spectrometry - methods</subject><subject>Substrate Specificity</subject><issn>1554-8929</issn><issn>1554-8937</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kElPwzAQhS0EomX5AxxQjlxS7DjxcoRSClJZJJarZTs2TZXExU4O-fe4aik3TjOa-d7TzAPgAsEJghm6ljropWlU5SZcQVhAcgDGqCjylHFMD_d9xkfgJIQVhDkmjB-DEUYUwZygMfictV9Va4w3ZTJdDo3r_LAO1XNinU-eZAjJ29rozrvGxE16K0ME70wXZ5VrE2eTV-86U7XJvB60C0MtN4szcGRlHcz5rp6Cj_vZ-_QhXbzMH6c3i1TiPOtSRpgiiHGVE2lLzSnlWhIuLYuXKxqvzTJEcGktJaTQinGmNLJZLiGFhhh8Cq62vmvvvnsTOtFUQZu6lq1xfRAZRjlEmDIa0WyLau9C8MaKta8a6QeBoNjkKf7yFLs8o-hy59-rxpR7yW-AEZhsgSgWK9f7Nr77n-MPna6EEQ</recordid><startdate>20191220</startdate><enddate>20191220</enddate><creator>Ramesh, Balakrishnan</creator><creator>Abnouf, Shaza</creator><creator>Mali, Sujina</creator><creator>Moree, Wilna J</creator><creator>Patil, Ujwal</creator><creator>Bark, Steven J</creator><creator>Varadarajan, Navin</creator><general>American Chemical Society</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>7X8</scope><orcidid>https://orcid.org/0000-0001-7524-8228</orcidid></search><sort><creationdate>20191220</creationdate><title>Engineered ChymotrypsiN for Mass Spectrometry-Based Detection of Protein Glycosylation</title><author>Ramesh, Balakrishnan ; Abnouf, Shaza ; Mali, Sujina ; Moree, Wilna J ; Patil, Ujwal ; Bark, Steven J ; Varadarajan, Navin</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a342t-868b6189b46afdc9779ca69af8554b736822163dff7665cb898bc1f24a070e6e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Chymotrypsin - genetics</topic><topic>Chymotrypsin - metabolism</topic><topic>Escherichia coli - genetics</topic><topic>Glycosylation</topic><topic>High-Throughput Screening Assays</topic><topic>Humans</topic><topic>Mass Spectrometry - methods</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ramesh, Balakrishnan</creatorcontrib><creatorcontrib>Abnouf, Shaza</creatorcontrib><creatorcontrib>Mali, Sujina</creatorcontrib><creatorcontrib>Moree, Wilna J</creatorcontrib><creatorcontrib>Patil, Ujwal</creatorcontrib><creatorcontrib>Bark, Steven J</creatorcontrib><creatorcontrib>Varadarajan, Navin</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>ACS chemical biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ramesh, Balakrishnan</au><au>Abnouf, Shaza</au><au>Mali, Sujina</au><au>Moree, Wilna J</au><au>Patil, Ujwal</au><au>Bark, Steven J</au><au>Varadarajan, Navin</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Engineered ChymotrypsiN for Mass Spectrometry-Based Detection of Protein Glycosylation</atitle><jtitle>ACS chemical biology</jtitle><addtitle>ACS Chem. Biol</addtitle><date>2019-12-20</date><risdate>2019</risdate><volume>14</volume><issue>12</issue><spage>2616</spage><epage>2628</epage><pages>2616-2628</pages><issn>1554-8929</issn><eissn>1554-8937</eissn><abstract>We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn-containing substrates. We confirmed that protein engineering did not compromise the stability of the enzyme by biophysical characterization. Comparison of wild-type ChyB and ChyB-Asn in profiling lysates of HEK293 cells demonstrated both qualitative and quantitative differences in the nature of the peptides and proteins identified by liquid chromatography and tandem mass spectrometry. ChyB-Asn enabled the identification of partially glycosylated Asn sites within a model glycoprotein and in the extracellular proteome of Jurkat T cells. ChymotrypsiN is a valuable addition to the toolkit of proteases to aid the mapping of N-linked glycosylation sites within proteins and proteomes.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>31710461</pmid><doi>10.1021/acschembio.9b00506</doi><tpages>13</tpages><orcidid>https://orcid.org/0000-0001-7524-8228</orcidid></addata></record> |
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| source | MEDLINE; American Chemical Society Journals |
| subjects | Chymotrypsin - genetics Chymotrypsin - metabolism Escherichia coli - genetics Glycosylation High-Throughput Screening Assays Humans Mass Spectrometry - methods Substrate Specificity |
| title | Engineered ChymotrypsiN for Mass Spectrometry-Based Detection of Protein Glycosylation |
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