Linked Life Data

16246579

Source:http://linkedlifedata.com/resource/pubmed/id/16246579

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
12
pubmed:dateCreated
2005-11-21
pubmed:abstractText
Chemical cross-linking of protein complexes has gained renewed interest in combination with mass spectrometric analysis of the reaction products as it allows a rapid mapping of protein interfaces, which is crucial for understanding protein/protein interactions. The identification of cross-linking products from the complex mixtures created after the cross-linking reaction, however, remains a daunting task. To facilitate the identification of cross-linking products, we explore the use of the commercially available biotinylated cross-linking reagent sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3'-dithiopropionate). This trifunctional cross-linker possesses one amine-reactive and one photo-reactive site and, additionally, allows an affinity-based enrichment of cross-linker containing species. As a model system, we chose the Ca(2+)-dependent complex between calmodulin and its target peptide M13, which represents a part of the C-terminal sequence of the skeletal muscle myosin light chain kinase. After the cross-linking reaction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and one-dimensional gel electrophoresis were employed to check for the extent of cross-linking product formation. The cross-linking reaction mixtures were subjected to tryptic in-solution digestion. Biotinylated peptides, e.g., peptides that had been modified by the cross-linker as well as cross-linked peptides, were enriched on monomeric avidin beads after several washing steps had been performed. Peptide mixtures were analyzed by MALDI-TOFMS, nano-high-performance liquid chromatography (HPLC)/nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS), and tandem MS. We demonstrate that an enrichment of cross-linker containing species allows a more efficient identification of interacting amino acid sequences in protein complexes. This strategy is expected to be especially beneficial for investigating large protein assemblies.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
1044-0305
pubmed:author
pubmed:issnType
Print
pubmed:volume
16
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1921-31
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Mapping protein interfaces by a trifunctional cross-linker combined with MALDI-TOF and ESI-FTICR mass spectrometry.
pubmed:affiliation
Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, Germany. sinz@chemie.uni-leipzig.de
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't