This chart shows the underlying motif distribution within 1044 nonredundant tryptic peptides with a carboxy-terminal aspartate derived from an LC-MS/MS experiment using HeLa cells treated with Staurosporine #9953 1μM, (3 hr; +) and immunoprecipitated with PTMScan® Cleaved Caspase Substrate Motif [DE(T/S/A)D] Immunoaffinity Beads. Within the same data set, the proportion of peptides with D in the -3 position is 36%, and the proportion of peptides with E in the -2 position is 53%.
The Motif Logo was generated from a PTMScan® LC-MS/MS experiment using 1044 nonredundant tryptic peptides with carboxy-terminal aspartates derived from human HeLa cells treated with Staurosporine #9953 1 μM for 3 hours to induce apoptosis. Peptides were immunoprecipitated with PTMScan® Cleaved Caspase Substrate Motif [DE(T/S/A)D] Immunoaffinity Beads. The logo represents the relative frequency of amino acids in each position leading up to the carboxy-terminal aspartate.
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.
Apoptosis is a physiological process resulting in a highly regulated, programmed form of cell death that is a normal part of growth and development in multicellular organisms. Aspartic acid-directed cysteine proteases, caspases, are central to the apoptotic mechanism (1). An intrinsic pathway initiates the apoptotic cascade from signals originating within the cell, such as DNA damage, and an extrinsic pathway initiates apoptosis in response to extracellular signals, like FasL. In either case, initiator caspases, such as caspase-8 and 9, begin cleaving downstream substrates that include the effector caspases, such as caspase-3, the most prominent executioner caspase (2,3). These effector caspases amplify the apoptotic cascade to target many critical proteins needed for normal cell function. Apart from its role in developmental biology, the regulation of apoptosis has broad implications for the study of cancer, autoimmune, and infectious diseases among others (4). In the human proteome, there are thousands of known or putative caspase cleavage sites and all are cleaved at an aspartic acid residue, or, in rare cases, a glutamic acid residue. The resultant fragments containing a carboxy-terminal aspartate generally have the XEXD motif with some variation (5). The PTMScan® Cleaved Caspase Substrate Motif [DE(T/S/A)D] Kit provides a unique and sensitive method for quantifying hundreds of cleaved caspase substrates, including caspase-3 and 7, NEDD1, HDAC3, MCM4, Notch1, eIF2B, raptor, Bid, Smad6, NFAT90, and PTEN among others.
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