SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides.
Mol Cell Proteomics
; 7(4): 661-71, 2008 Apr.
Article
en En
| MEDLINE
| ID: mdl-18039691
ABSTRACT
The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection, and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphorylated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide, the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy, SIMAC (sequential elution from IMAC), for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 mug of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a 3-fold increase in recovery of multiply phosphorylated peptides.
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Banco de datos:
MEDLINE
Asunto principal:
Fosfopéptidos
/
Proteómica
Límite:
Humans
Idioma:
En
Revista:
Mol Cell Proteomics
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BIOQUIMICA
Año:
2008
Tipo del documento:
Article
País de afiliación:
Dinamarca