Linked Life Data

20225856

Source:http://linkedlifedata.com/resource/pubmed/id/20225856

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
7
pubmed:dateCreated
2010-3-31
pubmed:abstractText
Many cellular processes are driven by protein complexes. Although the identification of protein components in such complexes has become almost a routine matter, accurate determination of their stoichiometry within a protein complex is still a challenge. We have established a method to determine the stoichiometries of protein complexes using absolute quantification (AQUA) with the help of synthetic standard peptides in combination with multiple reaction monitoring (MRM). Our approach is exemplified by the analysis of the human spliceosomal hPrp19/CDC5L complex, which consists of seven individual proteins and plays a crucial role in the assembly of the fully catalytically active spliceosome during pre-mRNA splicing. We evaluated several conditions for complete hydrolysis of the protein complex and found that the denaturing conditions under which hydrolysis is performed are absolutely crucial for accurately determining protein stoichiometries within this complex. In addition, we tested the suitability of different AQUA peptides and further compared different MS techniques to read out the relative signal intensities that were then used in absolute quantification. Our analyses revealed that dependent on the denaturing conditions different stoichiometries within the complex were obtained. The most consistent results were obtained by enzymatic hydrolysis in the presence of acetonitrile in combination with MRM.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1520-6882
pubmed:author
pubmed:issnType
Electronic
pubmed:day
1
pubmed:volume
82
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2784-96
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
Determination of protein stoichiometry within protein complexes using absolute quantification and multiple reaction monitoring.
pubmed:affiliation
Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't