This chart shows the distribution of underlying phospho-peptide motifs in a PhosphoScan® LC-MS/MS experiment using 754 nonredundant tryptic peptides generated from mitotic HeLa cells. HeLa cells were treated with thymidine, (2 mM, 18 hr) followed by Nocodazole #2190 (60 nM, 24 hr) and immunoprecipitated using PTMScan® Phospho-MAPK Substrate Motif [PXpTP] Immunoaffinity Beads.
The Motif Logo was generated from a PhosphoScan® LC-MS/MS experiment using 754 nonredundant tryptic peptides derived from mitotic HeLa cells that were treated with thymidine (2 mM, 18 hr) followed by Nocodazole #2190 (60 nM, 24 hr) and immunoprecipitated using PTMScan® Phospho-MAPK Substrate Motif [PXpTP] Immunoaffinity Beads. The Motif Logo demonstrates the relative abundance of amino acids in a given position within this data set.
Cells are lysed in a urea-containing buffer, cellular proteins are digested by proteases, and the resulting peptides are purified by reversed-phase solid-phase extraction. Peptides are then subjected to immunoaffinity purification using a PTMScan® Motif Antibody conjugated to protein A agarose beads. Unbound peptides are removed through washing, and the captured PTM-containing peptides are eluted with dilute acid. Reversed-phase purification is performed on microtips to desalt and separate peptides from antibody prior to concentrating the enriched peptides for LC-MS/MS analysis. CST recommends the use of PTMScan® IAP Buffer #9993 included in the kit. A detailed protocol and Limited Use License allowing the use of the patented PTMScan® method are included with the kit.
Antibody beads supplied in IAP buffer containing 50% glycerol. Store at -20°C. Do not aliquot the antibody.
PTMScan® Technology employs a proprietary methodology from Cell Signaling Technology (CST) for peptide enrichment by immunoprecipitation using a specific bead-conjugated antibody in conjunction with liquid chromatography (LC) tandem mass spectrometry (MS/MS) for quantitative profiling of post-translational modification (PTM) sites in cellular proteins. These include phosphorylation (PhosphoScan®), ubiquitination (UbiScan®), acetylation (AcetylScan®), and methylation (MethylScan®), among others. PTMScan® Technology enables researchers to isolate, identify, and quantitate large numbers of post-translationally modified cellular peptides with a high degree of specificity and sensitivity, providing a global overview of PTMs in cell and tissue samples without preconceived biases about where these modified sites occur. For more information on PTMScan® Proteomics Services, please visit www.cellsignal.com/common/content/content.jsp?id=ptmscan-services.
The MAPK and CDK families of serine/threonine protein kinases play important roles in proliferation and cell cycle control. These kinases phosphorylate threonine or serine followed by a proline residue (1-3). MAPK phosphorylates substrates with the consensus sequence PX(S/T)P, and CDKs phosphorylate substrates containing the consensus sequence (S/T)PXR/K. Cell Signaling Technology has developed antibodies that bind to phospho-threonine followed by proline, motifs PXS*/T*P and/or S*PXR/K, for use in the study and discovery of new MAPK and CDK substrates (4,5).
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