This chart shows the underlying motif distribution within 1111 nonredundant tryptic peptides derived from an LC-MS/MS experiment using Jurkat cells treated with Calyculin A #9902 and pervanadate and immunoprecipitated with PTMScan® Phospho-ATM/ATR Substrate Motif [pSQ] Immunoaffinity Beads. Within the same data set, phospho-serine in the central position constitutes 88% of the peptides, while phospho-threonine constitutes 12%. The most frequent submotif is [pSQG] with a 16% rate of occurrence relative to the total data set and 28% relative to the subset with the pSQ motif.
This Motif Logo was generated from a PhosphoScan® LC-MS/MS experiment using 1111 nonredundant tryptic peptides derived from Jurkat cells treated with Calyculin A #9902 and pervanadate and immunoprecipitated using PTMScan® Phospho-ATM/ATR Substrate Motif [pSQ] Immunoaffinity Beads. The PhosphoSitePlus® logo reflects the relative prevalence of an amino acid in each position relative to the phospho-serine background in the human proteome. Residues represented above the x-axis are enriched relative to their expected frequency in this background. For more information on motif analysis using PSP, please visit www.phosphosite.org.
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 (1). For more information on PTMScan® Proteomics Services, please visit www.cellsignal.com/common/content/content.jsp?id=ptmscan-services.
PhosphoScan® Technology employs a phospho-residue (Tyr, Ser, Thr) motif antibody for phospho-peptide immunoaffinity purification from cell extracts combined with LC tandem MS/MS to identify and quantify changes in phosphorylation levels (1). Ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) are related kinases that regulate cell cycle checkpoints and DNA repair (2). The identified substrates for ATM are p53, p95/NBS1, MDM2, Chk2, BRCA1, CtIP, 4E-BP1, and Chk1 (2,3). The consensus sequence for ATM/ATR substrates is [pS/pTQ]. Hydrophobic amino acids at positions -3 and -1, and negatively charged amino acids at position +1 are positive determinants for substrate recognition by these kinases. Positively charged residues surrounding the [pS/pTQ] are negative determinants for substrate phosphorylation (4). The complex phenotype of AT cells suggests that it likely has additional substrates (4). To better understand the kinase and identify substrates for ATM and the related kinase ATR, we have developed antibodies that recognize phosphorylated serine or threonine in the [pS/pTQ] motif.