Monitoring peptidase activities in complex proteomes by MALDI-TOF mass spectrometry
Measuring enzymatic activities in biological fluids is a form of activity-based proteomics and may be utilized as a means of developing disease biomarkers. Activity-based assays allow amplification of output signals, thus potentially visualizing low-abundant enzymes on a virtually transparent whole-...
Gespeichert in:
| Veröffentlicht in: | Nature protocols 2009-01, Vol.4 (8), p.1167-1183 |
|---|---|
| Hauptverfasser: | , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 1183 |
|---|---|
| container_issue | 8 |
| container_start_page | 1167 |
| container_title | Nature protocols |
| container_volume | 4 |
| creator | Villanueva, Josep Nazarian, Arpi Lawlor, Kevin Tempst, Paul |
| description | Measuring enzymatic activities in biological fluids is a form of activity-based proteomics and may be utilized as a means of developing disease biomarkers. Activity-based assays allow amplification of output signals, thus potentially visualizing low-abundant enzymes on a virtually transparent whole-proteome background. The protocol presented here describes a semiquantitative
in vitro
assay of proteolytic activities in complex proteomes by monitoring breakdown of designer peptide substrates using robotic extraction and a matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometric readout. Relative quantitation of the peptide metabolites is carried out by comparison with spiked internal standards, followed by statistical analysis of the resulting mini-peptidome. Partial automation provides reproducibility and throughput essential for comparing large sample sets. The approach may be used for diagnostic or predictive purposes and it enables profiling of 96 samples in 30 h. It could be tailored to many diagnostic and pharmaco-dynamic purposes as a readout of catalytic and metabolic activities in body fluids or tissues. |
| doi_str_mv | 10.1038/nprot.2009.88 |
| format | Article |
| fullrecord | <record><control><sourceid>gale_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_2760592</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><galeid>A205985207</galeid><sourcerecordid>A205985207</sourcerecordid><originalsourceid>FETCH-LOGICAL-c656t-24d72ac82c5ebcf667c45cf5c6f8233f3cc2a9b6585a28a551d7301a9b58402f3</originalsourceid><addsrcrecordid>eNp1kttrFDEUxgdR7EUffZUBH0Rk1lwml3kpLGurC1sLdsXHkM1k1pSZZEwypfvfm2kXtyOVPCSc88t3OB9flr2BYAYB5p9s712cIQCqGefPsmPICCgQq6rn9--yQJBXR9lJCDcAlAxT9jI7ghWFjHN-nF1fOmui88Zu81730dQy6FyqaG5NNDrkxubKdX2r7_JxknZdKm52-eV89XlZrK8u8k6GkIdeq-hTM_rdq-xFI9ugX-_v0-zHxfl68bVYXX1ZLuarQlFCY4HKmiGpOFJEb1RDKVMlUQ1RtOEI4wYrhWS1oYQTibgkBNYMA5hKhJcANfg0O3vQ7YdNp2ulbfSyFb03nfQ74aQR0441v8TW3QrEKCAVSgLv9wLe_R50iKIzQem2lVa7IQiGcfKrKkfy3T_kjRu8TdsJiBHFsASEH6itbLUwtnFprBo1xRyliZwgwBI1e4JKp9adUc7qxqT65MOHyYfERH0Xt3IIQSyvv0_Zj_9n5-ufi29TuniglXcheN38NQ8CMcZL3MdLjPESfFzw7WPHD_Q-T4fdQj9GSvtHPj2p-Aff-Nl8</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1326314058</pqid></control><display><type>article</type><title>Monitoring peptidase activities in complex proteomes by MALDI-TOF mass spectrometry</title><source>MEDLINE</source><source>SpringerLink Journals</source><source>Nature Journals Online</source><creator>Villanueva, Josep ; Nazarian, Arpi ; Lawlor, Kevin ; Tempst, Paul</creator><creatorcontrib>Villanueva, Josep ; Nazarian, Arpi ; Lawlor, Kevin ; Tempst, Paul</creatorcontrib><description>Measuring enzymatic activities in biological fluids is a form of activity-based proteomics and may be utilized as a means of developing disease biomarkers. Activity-based assays allow amplification of output signals, thus potentially visualizing low-abundant enzymes on a virtually transparent whole-proteome background. The protocol presented here describes a semiquantitative
in vitro
assay of proteolytic activities in complex proteomes by monitoring breakdown of designer peptide substrates using robotic extraction and a matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometric readout. Relative quantitation of the peptide metabolites is carried out by comparison with spiked internal standards, followed by statistical analysis of the resulting mini-peptidome. Partial automation provides reproducibility and throughput essential for comparing large sample sets. The approach may be used for diagnostic or predictive purposes and it enables profiling of 96 samples in 30 h. It could be tailored to many diagnostic and pharmaco-dynamic purposes as a readout of catalytic and metabolic activities in body fluids or tissues.</description><identifier>ISSN: 1754-2189</identifier><identifier>EISSN: 1750-2799</identifier><identifier>DOI: 10.1038/nprot.2009.88</identifier><identifier>PMID: 19617888</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>Analytical Chemistry ; Automation ; Biological Techniques ; Biomarkers ; Biomedical and Life Sciences ; Cancer ; Computational Biology/Bioinformatics ; Desorption ; Enzymatic activity ; Enzymes ; Humans ; Ionization ; Life Sciences ; Mass spectrometry ; Metabolites ; Microarrays ; Organic Chemistry ; Peptide Hydrolases - metabolism ; Peptides ; Proteases ; Proteins ; Proteome ; Proteomics ; Proteomics - methods ; protocol ; Robotics ; Scientific imaging ; Software ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods ; Statistical analysis</subject><ispartof>Nature protocols, 2009-01, Vol.4 (8), p.1167-1183</ispartof><rights>Springer Nature Limited 2009</rights><rights>COPYRIGHT 2009 Nature Publishing Group</rights><rights>Copyright Nature Publishing Group Aug 2009</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c656t-24d72ac82c5ebcf667c45cf5c6f8233f3cc2a9b6585a28a551d7301a9b58402f3</citedby><cites>FETCH-LOGICAL-c656t-24d72ac82c5ebcf667c45cf5c6f8233f3cc2a9b6585a28a551d7301a9b58402f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/nprot.2009.88$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/nprot.2009.88$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,314,777,781,882,27905,27906,41469,42538,51300</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19617888$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Villanueva, Josep</creatorcontrib><creatorcontrib>Nazarian, Arpi</creatorcontrib><creatorcontrib>Lawlor, Kevin</creatorcontrib><creatorcontrib>Tempst, Paul</creatorcontrib><title>Monitoring peptidase activities in complex proteomes by MALDI-TOF mass spectrometry</title><title>Nature protocols</title><addtitle>Nat Protoc</addtitle><addtitle>Nat Protoc</addtitle><description>Measuring enzymatic activities in biological fluids is a form of activity-based proteomics and may be utilized as a means of developing disease biomarkers. Activity-based assays allow amplification of output signals, thus potentially visualizing low-abundant enzymes on a virtually transparent whole-proteome background. The protocol presented here describes a semiquantitative
in vitro
assay of proteolytic activities in complex proteomes by monitoring breakdown of designer peptide substrates using robotic extraction and a matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometric readout. Relative quantitation of the peptide metabolites is carried out by comparison with spiked internal standards, followed by statistical analysis of the resulting mini-peptidome. Partial automation provides reproducibility and throughput essential for comparing large sample sets. The approach may be used for diagnostic or predictive purposes and it enables profiling of 96 samples in 30 h. It could be tailored to many diagnostic and pharmaco-dynamic purposes as a readout of catalytic and metabolic activities in body fluids or tissues.</description><subject>Analytical Chemistry</subject><subject>Automation</subject><subject>Biological Techniques</subject><subject>Biomarkers</subject><subject>Biomedical and Life Sciences</subject><subject>Cancer</subject><subject>Computational Biology/Bioinformatics</subject><subject>Desorption</subject><subject>Enzymatic activity</subject><subject>Enzymes</subject><subject>Humans</subject><subject>Ionization</subject><subject>Life Sciences</subject><subject>Mass spectrometry</subject><subject>Metabolites</subject><subject>Microarrays</subject><subject>Organic Chemistry</subject><subject>Peptide Hydrolases - metabolism</subject><subject>Peptides</subject><subject>Proteases</subject><subject>Proteins</subject><subject>Proteome</subject><subject>Proteomics</subject><subject>Proteomics - methods</subject><subject>protocol</subject><subject>Robotics</subject><subject>Scientific imaging</subject><subject>Software</subject><subject>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods</subject><subject>Statistical analysis</subject><issn>1754-2189</issn><issn>1750-2799</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp1kttrFDEUxgdR7EUffZUBH0Rk1lwml3kpLGurC1sLdsXHkM1k1pSZZEwypfvfm2kXtyOVPCSc88t3OB9flr2BYAYB5p9s712cIQCqGefPsmPICCgQq6rn9--yQJBXR9lJCDcAlAxT9jI7ghWFjHN-nF1fOmui88Zu81730dQy6FyqaG5NNDrkxubKdX2r7_JxknZdKm52-eV89XlZrK8u8k6GkIdeq-hTM_rdq-xFI9ugX-_v0-zHxfl68bVYXX1ZLuarQlFCY4HKmiGpOFJEb1RDKVMlUQ1RtOEI4wYrhWS1oYQTibgkBNYMA5hKhJcANfg0O3vQ7YdNp2ulbfSyFb03nfQ74aQR0441v8TW3QrEKCAVSgLv9wLe_R50iKIzQem2lVa7IQiGcfKrKkfy3T_kjRu8TdsJiBHFsASEH6itbLUwtnFprBo1xRyliZwgwBI1e4JKp9adUc7qxqT65MOHyYfERH0Xt3IIQSyvv0_Zj_9n5-ufi29TuniglXcheN38NQ8CMcZL3MdLjPESfFzw7WPHD_Q-T4fdQj9GSvtHPj2p-Aff-Nl8</recordid><startdate>20090101</startdate><enddate>20090101</enddate><creator>Villanueva, Josep</creator><creator>Nazarian, Arpi</creator><creator>Lawlor, Kevin</creator><creator>Tempst, Paul</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>ATWCN</scope><scope>ISR</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>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20090101</creationdate><title>Monitoring peptidase activities in complex proteomes by MALDI-TOF mass spectrometry</title><author>Villanueva, Josep ; Nazarian, Arpi ; Lawlor, Kevin ; Tempst, Paul</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c656t-24d72ac82c5ebcf667c45cf5c6f8233f3cc2a9b6585a28a551d7301a9b58402f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>Analytical Chemistry</topic><topic>Automation</topic><topic>Biological Techniques</topic><topic>Biomarkers</topic><topic>Biomedical and Life Sciences</topic><topic>Cancer</topic><topic>Computational Biology/Bioinformatics</topic><topic>Desorption</topic><topic>Enzymatic activity</topic><topic>Enzymes</topic><topic>Humans</topic><topic>Ionization</topic><topic>Life Sciences</topic><topic>Mass spectrometry</topic><topic>Metabolites</topic><topic>Microarrays</topic><topic>Organic Chemistry</topic><topic>Peptide Hydrolases - metabolism</topic><topic>Peptides</topic><topic>Proteases</topic><topic>Proteins</topic><topic>Proteome</topic><topic>Proteomics</topic><topic>Proteomics - methods</topic><topic>protocol</topic><topic>Robotics</topic><topic>Scientific imaging</topic><topic>Software</topic><topic>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods</topic><topic>Statistical analysis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Villanueva, Josep</creatorcontrib><creatorcontrib>Nazarian, Arpi</creatorcontrib><creatorcontrib>Lawlor, Kevin</creatorcontrib><creatorcontrib>Tempst, Paul</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Middle School</collection><collection>Gale In Context: Science</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 (ProQuest)</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 One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</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>Villanueva, Josep</au><au>Nazarian, Arpi</au><au>Lawlor, Kevin</au><au>Tempst, Paul</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Monitoring peptidase activities in complex proteomes by MALDI-TOF mass spectrometry</atitle><jtitle>Nature protocols</jtitle><stitle>Nat Protoc</stitle><addtitle>Nat Protoc</addtitle><date>2009-01-01</date><risdate>2009</risdate><volume>4</volume><issue>8</issue><spage>1167</spage><epage>1183</epage><pages>1167-1183</pages><issn>1754-2189</issn><eissn>1750-2799</eissn><abstract>Measuring enzymatic activities in biological fluids is a form of activity-based proteomics and may be utilized as a means of developing disease biomarkers. Activity-based assays allow amplification of output signals, thus potentially visualizing low-abundant enzymes on a virtually transparent whole-proteome background. The protocol presented here describes a semiquantitative
in vitro
assay of proteolytic activities in complex proteomes by monitoring breakdown of designer peptide substrates using robotic extraction and a matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometric readout. Relative quantitation of the peptide metabolites is carried out by comparison with spiked internal standards, followed by statistical analysis of the resulting mini-peptidome. Partial automation provides reproducibility and throughput essential for comparing large sample sets. The approach may be used for diagnostic or predictive purposes and it enables profiling of 96 samples in 30 h. It could be tailored to many diagnostic and pharmaco-dynamic purposes as a readout of catalytic and metabolic activities in body fluids or tissues.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>19617888</pmid><doi>10.1038/nprot.2009.88</doi><tpages>17</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1754-2189 |
| ispartof | Nature protocols, 2009-01, Vol.4 (8), p.1167-1183 |
| issn | 1754-2189 1750-2799 |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_2760592 |
| source | MEDLINE; SpringerLink Journals; Nature Journals Online |
| subjects | Analytical Chemistry Automation Biological Techniques Biomarkers Biomedical and Life Sciences Cancer Computational Biology/Bioinformatics Desorption Enzymatic activity Enzymes Humans Ionization Life Sciences Mass spectrometry Metabolites Microarrays Organic Chemistry Peptide Hydrolases - metabolism Peptides Proteases Proteins Proteome Proteomics Proteomics - methods protocol Robotics Scientific imaging Software Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods Statistical analysis |
| title | Monitoring peptidase activities in complex proteomes by MALDI-TOF mass spectrometry |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-20T05%3A21%3A04IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Monitoring%20peptidase%20activities%20in%20complex%20proteomes%20by%20MALDI-TOF%20mass%20spectrometry&rft.jtitle=Nature%20protocols&rft.au=Villanueva,%20Josep&rft.date=2009-01-01&rft.volume=4&rft.issue=8&rft.spage=1167&rft.epage=1183&rft.pages=1167-1183&rft.issn=1754-2189&rft.eissn=1750-2799&rft_id=info:doi/10.1038/nprot.2009.88&rft_dat=%3Cgale_pubme%3EA205985207%3C/gale_pubme%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1326314058&rft_id=info:pmid/19617888&rft_galeid=A205985207&rfr_iscdi=true |