Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
7
pubmed:dateCreated
2010-7-2
pubmed:abstractText
Immobilized metal ion affinity chromatography (IMAC) is widely used for phosphopeptide enrichment. However, the robustness, efficiency, and specificity of this technique in large-scale phosphoproteomics studies are still disputed. In this study, we first compared three widely used IMAC materials under three different conditions. Fe(III)-nitrilotriacetic acid (NTA) IMAC resin was chosen due to its superior performance in all tests. We further investigated the solution ionization efficiency change of the phosphoryl group and carboxylic group in different acetonitrile-water solutions and observed that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol was established using Fe(III)-NTA IMAC resin and it proved to be highly selective in the phosphopeptide enrichment of a highly diluted standard sample (1:1000) prior to MALDI MS analysis. We also observed that a higher iron purity led to an increased IMAC enrichment efficiency. The optimized method was then adapted to phosphoproteome analyses of cell lysates of high protein complexity. From either 20 microg of mouse sample or 50 microg of Drosophila melanogaster sample, more than 1000 phosphorylation sites were identified in each study using IMAC-IMAC and LC-MS/MS. We demonstrate efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC-IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/Acetonitriles, http://linkedlifedata.com/resource/pubmed/chemical/Cell Extracts, http://linkedlifedata.com/resource/pubmed/chemical/Complex Mixtures, http://linkedlifedata.com/resource/pubmed/chemical/Drosophila Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Ferric Compounds, http://linkedlifedata.com/resource/pubmed/chemical/Nitrilotriacetic Acid, http://linkedlifedata.com/resource/pubmed/chemical/Peptide Fragments, http://linkedlifedata.com/resource/pubmed/chemical/Phosphopeptides, http://linkedlifedata.com/resource/pubmed/chemical/Phosphoproteins, http://linkedlifedata.com/resource/pubmed/chemical/Trypsin, http://linkedlifedata.com/resource/pubmed/chemical/acetonitrile, http://linkedlifedata.com/resource/pubmed/chemical/ferric nitrilotriacetate
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
1535-3907
pubmed:author
pubmed:issnType
Electronic
pubmed:day
2
pubmed:volume
9
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3561-73
pubmed:meshHeading
pubmed-meshheading:20450229-Acetonitriles, pubmed-meshheading:20450229-Animals, pubmed-meshheading:20450229-Cattle, pubmed-meshheading:20450229-Cell Extracts, pubmed-meshheading:20450229-Chromatography, Affinity, pubmed-meshheading:20450229-Complex Mixtures, pubmed-meshheading:20450229-Databases, Protein, pubmed-meshheading:20450229-Drosophila Proteins, pubmed-meshheading:20450229-Ferric Compounds, pubmed-meshheading:20450229-Hydrogen-Ion Concentration, pubmed-meshheading:20450229-Lymphocytes, pubmed-meshheading:20450229-Mice, pubmed-meshheading:20450229-Nitrilotriacetic Acid, pubmed-meshheading:20450229-Peptide Fragments, pubmed-meshheading:20450229-Phosphopeptides, pubmed-meshheading:20450229-Phosphoproteins, pubmed-meshheading:20450229-Tandem Mass Spectrometry, pubmed-meshheading:20450229-Trypsin
pubmed:year
2010
pubmed:articleTitle
Optimized IMAC-IMAC protocol for phosphopeptide recovery from complex biological samples.
pubmed:affiliation
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't