SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides.

@article{citeulike:3070706, 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.}, address = {Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.}, author = {Thingholm, T. E. and Jensen, O. N. and Robinson, P. J. and Larsen, M. R. }, citeulike-article-id = {3070706}, doi = {10.1074/mcp.M700362-MCP200}, issn = {1535-9484}, journal = {Molecular \& cellular proteomics : MCP}, keywords = {elution, from, imac, monophosphorylated, multiply, peptides, phosphoproteomics, phosphorylated, rapid, separation, sequential, simac, strategy}, month = {April}, number = {4}, pages = {661--671}, posted-at = {2008-08-01 09:43:27}, priority = {2}, title = {SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides.}, url = {http://dx.doi.org/10.1074/mcp.M700362-MCP200}, volume = {7}, year = {2008} }

TY - JOUR ID - citeulike:3070706 L3 - citeulike-article-id:3070706 TI - SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides. JF - Molecular & cellular proteomics : MCP VL - 7 IS - 4 SP - 661 EP - 671 AD - Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark. SN - 1535-9484 N2 - 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. KW - elution KW - from KW - imac KW - monophosphorylated KW - multiply KW - peptides KW - phosphoproteomics KW - phosphorylated KW - rapid KW - separation KW - sequential KW - simac KW - strategy AU - Thingholm, TE AU - Jensen, ON AU - Robinson, PJ AU - Larsen, MR PY - 2008/04// UR - http://dx.doi.org/10.1074/mcp.M700362-MCP200 ER -