Enrichment and sequencing of phosphopeptides on indium tin oxide coated glass slides.
Identifieur interne : 001444 ( Main/Exploration ); précédent : 001443; suivant : 001445Enrichment and sequencing of phosphopeptides on indium tin oxide coated glass slides.
Auteurs : RBID : pubmed:21523302English descriptors
- KwdEn :
- Amino Acid Sequence, Animals, COS Cells, Caseins (chemistry), Cercopithecus aethiops, Cyclic AMP-Dependent Protein Kinases (chemistry), Glass, Limit of Detection, NFATC Transcription Factors (biosynthesis), NFATC Transcription Factors (chemistry), Phosphoproteins (chemistry), Phosphoproteins (isolation & purification), Phosphorus Radioisotopes, Phosphorylation, Recombinant Proteins (biosynthesis), Recombinant Proteins (chemistry), Sequence Analysis, Protein, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Tandem Mass Spectrometry, Tin Compounds (chemistry).
- MESH :
- chemical , biosynthesis : NFATC Transcription Factors, Recombinant Proteins.
- chemical , chemistry : Caseins, Cyclic AMP-Dependent Protein Kinases, NFATC Transcription Factors, Phosphoproteins, Recombinant Proteins, Tin Compounds.
- chemical , isolation & purification : Phosphoproteins.
- Amino Acid Sequence, Animals, COS Cells, Cercopithecus aethiops, Glass, Limit of Detection, Phosphorus Radioisotopes, Phosphorylation, Sequence Analysis, Protein, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Tandem Mass Spectrometry.
Abstract
Unambiguous identification of phosphorylation sites is of premier importance to biologists, who seek to understand the role of phosphorylation from the perspective of site-specific control of biological phenomena. Despite this widely asked and highly specific information, many methods developed are aimed at analysis of complete proteomes, indeed even phospho-proteomes, surpassing the basic requests of many biologists. We have therefore further developed a simple method that specifically deals with the analysis of multiple phosphorylation sites on singular proteins or small collections of proteins. With this method, the whole purification process, from sample application to MALDI-MS analysis, can be performed on commercially available indium tin oxide (ITO) coated glass slides. We show that fifteen (15) samples can be purified within one hour, and that low femtomole sensitivity can be achieved. This limit of identification is demonstrated by the successful MS/MS-based identification of 6 fmol of monophosphopeptide from β-casein. We demonstrate that the method can be applied for identifying phosphorylation sites from recombinant and cell-derived biological protein samples. Since ITO-coated glass slides are inexpensive and available from several suppliers the method is readily and inexpensively available to other researchers. Taken together, the presented protocols and materials render this method as an extremely fast and sensitive phosphopeptide identification protocol that should aid biologists in discovery and validation of phosphorylation sites.
DOI: 10.1039/c0mb00269k
PubMed: 21523302
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Enrichment and sequencing of phosphopeptides on indium tin oxide coated glass slides.</title><author><name sortKey="Kouvonen, Petri" uniqKey="Kouvonen P">Petri Kouvonen</name><affiliation wicri:level="1"><nlm:affiliation>University of Turku, Centre for Biotechnology, Turku, Finland.</nlm:affiliation><country xml:lang="fr">Finlande</country><wicri:regionArea>University of Turku, Centre for Biotechnology, Turku</wicri:regionArea></affiliation></author><author><name sortKey="Rainio, Eeva Marja" uniqKey="Rainio E">Eeva-Marja Rainio</name></author><author><name sortKey="Suni, Veronika" uniqKey="Suni V">Veronika Suni</name></author><author><name sortKey="Koskinen, P Ivi" uniqKey="Koskinen P">Päivi Koskinen</name></author><author><name sortKey="Corthals, Garry L" uniqKey="Corthals G">Garry L Corthals</name></author></titleStmt><publicationStmt><date when="2011">2011</date><idno type="doi">10.1039/c0mb00269k</idno><idno type="RBID">pubmed:21523302</idno><idno type="pmid">21523302</idno><idno type="wicri:Area/Main/Corpus">001405</idno><idno type="wicri:Area/Main/Curation">001405</idno><idno type="wicri:Area/Main/Exploration">001444</idno></publicationStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amino Acid Sequence</term><term>Animals</term><term>COS Cells</term><term>Caseins (chemistry)</term><term>Cercopithecus aethiops</term><term>Cyclic AMP-Dependent Protein Kinases (chemistry)</term><term>Glass</term><term>Limit of Detection</term><term>NFATC Transcription Factors (biosynthesis)</term><term>NFATC Transcription Factors (chemistry)</term><term>Phosphoproteins (chemistry)</term><term>Phosphoproteins (isolation & purification)</term><term>Phosphorus Radioisotopes</term><term>Phosphorylation</term><term>Recombinant Proteins (biosynthesis)</term><term>Recombinant Proteins (chemistry)</term><term>Sequence Analysis, Protein</term><term>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</term><term>Tandem Mass Spectrometry</term><term>Tin Compounds (chemistry)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>NFATC Transcription Factors</term><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Caseins</term><term>Cyclic AMP-Dependent Protein Kinases</term><term>NFATC Transcription Factors</term><term>Phosphoproteins</term><term>Recombinant Proteins</term><term>Tin Compounds</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>Phosphoproteins</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Amino Acid Sequence</term><term>Animals</term><term>COS Cells</term><term>Cercopithecus aethiops</term><term>Glass</term><term>Limit of Detection</term><term>Phosphorus Radioisotopes</term><term>Phosphorylation</term><term>Sequence Analysis, Protein</term><term>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</term><term>Tandem Mass Spectrometry</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Unambiguous identification of phosphorylation sites is of premier importance to biologists, who seek to understand the role of phosphorylation from the perspective of site-specific control of biological phenomena. Despite this widely asked and highly specific information, many methods developed are aimed at analysis of complete proteomes, indeed even phospho-proteomes, surpassing the basic requests of many biologists. We have therefore further developed a simple method that specifically deals with the analysis of multiple phosphorylation sites on singular proteins or small collections of proteins. With this method, the whole purification process, from sample application to MALDI-MS analysis, can be performed on commercially available indium tin oxide (ITO) coated glass slides. We show that fifteen (15) samples can be purified within one hour, and that low femtomole sensitivity can be achieved. This limit of identification is demonstrated by the successful MS/MS-based identification of 6 fmol of monophosphopeptide from β-casein. We demonstrate that the method can be applied for identifying phosphorylation sites from recombinant and cell-derived biological protein samples. Since ITO-coated glass slides are inexpensive and available from several suppliers the method is readily and inexpensively available to other researchers. Taken together, the presented protocols and materials render this method as an extremely fast and sensitive phosphopeptide identification protocol that should aid biologists in discovery and validation of phosphorylation sites.</div></front></TEI><pubmed><MedlineCitation Owner="NLM" Status="MEDLINE"><PMID Version="1">21523302</PMID><DateCreated><Year>2011</Year><Month>05</Month><Day>16</Day></DateCreated><DateCompleted><Year>2011</Year><Month>09</Month><Day>07</Day></DateCompleted><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1742-2051</ISSN><JournalIssue CitedMedium="Internet"><Volume>7</Volume><Issue>6</Issue><PubDate><Year>2011</Year><Month>Jun</Month></PubDate></JournalIssue><Title>Molecular bioSystems</Title><ISOAbbreviation>Mol Biosyst</ISOAbbreviation></Journal><ArticleTitle>Enrichment and sequencing of phosphopeptides on indium tin oxide coated glass slides.</ArticleTitle><Pagination><MedlinePgn>1828-37</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1039/c0mb00269k</ELocationID><Abstract><AbstractText>Unambiguous identification of phosphorylation sites is of premier importance to biologists, who seek to understand the role of phosphorylation from the perspective of site-specific control of biological phenomena. Despite this widely asked and highly specific information, many methods developed are aimed at analysis of complete proteomes, indeed even phospho-proteomes, surpassing the basic requests of many biologists. We have therefore further developed a simple method that specifically deals with the analysis of multiple phosphorylation sites on singular proteins or small collections of proteins. With this method, the whole purification process, from sample application to MALDI-MS analysis, can be performed on commercially available indium tin oxide (ITO) coated glass slides. We show that fifteen (15) samples can be purified within one hour, and that low femtomole sensitivity can be achieved. This limit of identification is demonstrated by the successful MS/MS-based identification of 6 fmol of monophosphopeptide from β-casein. We demonstrate that the method can be applied for identifying phosphorylation sites from recombinant and cell-derived biological protein samples. Since ITO-coated glass slides are inexpensive and available from several suppliers the method is readily and inexpensively available to other researchers. Taken together, the presented protocols and materials render this method as an extremely fast and sensitive phosphopeptide identification protocol that should aid biologists in discovery and validation of phosphorylation sites.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Kouvonen</LastName><ForeName>Petri</ForeName><Initials>P</Initials><Affiliation>University of Turku, Centre for Biotechnology, Turku, Finland.</Affiliation></Author><Author ValidYN="Y"><LastName>Rainio</LastName><ForeName>Eeva-Marja</ForeName><Initials>EM</Initials></Author><Author ValidYN="Y"><LastName>Suni</LastName><ForeName>Veronika</ForeName><Initials>V</Initials></Author><Author ValidYN="Y"><LastName>Koskinen</LastName><ForeName>Päivi</ForeName><Initials>P</Initials></Author><Author ValidYN="Y"><LastName>Corthals</LastName><ForeName>Garry L</ForeName><Initials>GL</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType>Journal Article</PublicationType><PublicationType>Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2011</Year><Month>04</Month><Day>27</Day></ArticleDate></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Mol Biosyst</MedlineTA><NlmUniqueID>101251620</NlmUniqueID><ISSNLinking>1742-2051</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance>Caseins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance>NFATC Transcription Factors</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance>Phosphoproteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance>Phosphorus Radioisotopes</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance>Recombinant Proteins</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance>Tin Compounds</NameOfSubstance></Chemical><Chemical><RegistryNumber>71243-84-0</RegistryNumber><NameOfSubstance>indium tin oxide</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 2.7.11.11</RegistryNumber><NameOfSubstance>Cyclic AMP-Dependent Protein Kinases</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName MajorTopicYN="N">Amino Acid Sequence</DescriptorName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Animals</DescriptorName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">COS Cells</DescriptorName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Caseins</DescriptorName><QualifierName MajorTopicYN="N">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Cercopithecus aethiops</DescriptorName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Cyclic AMP-Dependent Protein Kinases</DescriptorName><QualifierName MajorTopicYN="N">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Glass</DescriptorName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Limit of Detection</DescriptorName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">NFATC Transcription Factors</DescriptorName><QualifierName MajorTopicYN="N">biosynthesis</QualifierName><QualifierName MajorTopicYN="N">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Phosphoproteins</DescriptorName><QualifierName MajorTopicYN="Y">chemistry</QualifierName><QualifierName MajorTopicYN="N">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Phosphorus Radioisotopes</DescriptorName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Phosphorylation</DescriptorName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Recombinant Proteins</DescriptorName><QualifierName MajorTopicYN="N">biosynthesis</QualifierName><QualifierName MajorTopicYN="N">chemistry</QualifierName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Sequence Analysis, Protein</DescriptorName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</DescriptorName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Tandem Mass Spectrometry</DescriptorName></MeshHeading><MeshHeading><DescriptorName MajorTopicYN="N">Tin Compounds</DescriptorName><QualifierName MajorTopicYN="Y">chemistry</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="aheadofprint"><Year>2011</Year><Month>4</Month><Day>27</Day></PubMedPubDate><PubMedPubDate PubStatus="epublish"><Year>2011</Year><Month>6</Month><Day>1</Day></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2011</Year><Month>4</Month><Day>28</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2011</Year><Month>4</Month><Day>28</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2011</Year><Month>9</Month><Day>8</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="doi">10.1039/c0mb00269k</ArticleId><ArticleId IdType="pubmed">21523302</ArticleId></ArticleIdList></PubmedData></pubmed></record>Pour manipuler ce document sous Unix (Dilib)
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