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Molecular & Cellular Proteomics 7:661-671, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Special Issue: 8th International Symposium On Mass Spectrometry In The Life Sciences

SIMAC (Sequential Elution from IMAC), a Phosphoproteomics Strategy for the Rapid Separation of Monophosphorylated from Multiply Phosphorylated Peptides^*,S

Tine E. Thingholm, Ole N. Jensen, Phillip J. Robinson and Martin R. Larsen^,¶

From the Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark and Cell Signaling Unit, Children's Medical Research Institute, The University of Sydney, Westmead, New South Wales 2145, Australia

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 µg 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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