Source:http://linkedlifedata.com/resource/pubmed/id/16750914
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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
8
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pubmed:dateCreated |
2006-7-31
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pubmed:abstractText |
For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an "IQ-like motif." Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d0 and d4) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central alpha-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural data of protein assemblies within a few days.
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pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical | |
pubmed:status |
MEDLINE
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pubmed:month |
Aug
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pubmed:issn |
1044-0305
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pubmed:author |
pubmed-author:Beck-SickingerAnnette GAG,
pubmed-author:CooperDermot M FDM,
pubmed-author:HaackMichaelM,
pubmed-author:IhlingChristianC,
pubmed-author:KalkhofStefanS,
pubmed-author:MechtlerKarlK,
pubmed-author:SchmidtAndreasA,
pubmed-author:SchulzDaniela MDM,
pubmed-author:SinzAndreaA,
pubmed-author:StinglChristophC
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pubmed:issnType |
Print
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pubmed:volume |
17
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
1100-13
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pubmed:meshHeading |
pubmed-meshheading:16750914-Adenylate Cyclase,
pubmed-meshheading:16750914-Animals,
pubmed-meshheading:16750914-Binding Sites,
pubmed-meshheading:16750914-Brain Chemistry,
pubmed-meshheading:16750914-Calmodulin,
pubmed-meshheading:16750914-Cross-Linking Reagents,
pubmed-meshheading:16750914-Cyclotrons,
pubmed-meshheading:16750914-Isotope Labeling,
pubmed-meshheading:16750914-Mass Spectrometry,
pubmed-meshheading:16750914-Peptide Mapping,
pubmed-meshheading:16750914-Protein Binding,
pubmed-meshheading:16750914-Sequence Analysis, Protein,
pubmed-meshheading:16750914-Spectroscopy, Fourier Transform Infrared,
pubmed-meshheading:16750914-Swine
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pubmed:year |
2006
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pubmed:articleTitle |
Isotope-labeled cross-linkers and Fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex.
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pubmed:affiliation |
Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, Leipzig, Germany.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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