The chart shows the relative category distribution of proteins with succinylated lysine residues identified from peptides generated from a PTMScan® LC-MS/MS experiment of mouse liver tissue using PTMScan® Succinyl-Lysine Motif [Succ-K] Immunoaffinity Beads.
The Motif Logo was generated from a PTMScan® LC-MS/MS experiment using 1467 nonredundant tryptic peptides derived from mouse liver immunoprecipitated with PTMScan® Succinyl-Lysine Motif [Succ-K] Immunoaffinity Beads. The logo represents the relative frequency of amino acids in each position surrounding the central succinylated lysine residue.
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.
Lysine is subject to a wide array of regulatory post-translational modifications due to its positively charged ε-amino group side chain. The most prevalent of these are ubiquitination and acetylation, which are highly conserved among prokaryotes and eukaryotes (1,2). Acyl group transfer from the metabolic intermediates acetyl-, succinyl-, malonyl-, glutaryl-, butyryl-, propionyl-, and crotonyl-CoA all neutralize lysine’s positive charge and confer structural alterations affecting substrate protein function. Lysine acetylation is catalyzed by histone acetyltransferases, HATs, using acetyl-CoA as a cofactor (3,4). Deacylation is mediated by histone deacetylases, HDACs 1-11, and NAD-dependent Sirtuins 1-7. Some sirtuins have little to no deacetylase activity, suggesting that they are better suited for other acyl lysine substrates (5).
Sirt 5 is a predominantly mitochondrial desuccinylase and demalonylase (5,6). In the absence of a known succinyltransferase, succinylation is likely driven by the concentration of succinyl-CoA and intracellular pH and is subject to metabolic fluctuations (7,8). Protein succinylation is especially prevalent among mitochondrial metabolic proteins and bacteria, further solidifying the evolutionary link between mitochondria and prokaryotes. It often occurs at lysine residues that are alternatively acetylated or ubiquitinated. More than a thousand lysine succinylation sites were identified on hundreds of proteins including glutamate dehydrogenase (15 sites), malate dehydrogenase, citrate synthase, carbamoyl phosphate synthase 1, and histone proteins (9).
AcetylScan is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
MethylScan is a trademark of Cell Signaling Technology, Inc.
PhosphoScan is a trademark of Cell Signaling Technology, Inc.
PTMScan is a trademark of Cell Signaling Technology, Inc.
UbiScan is a trademark of Cell Signaling Technology, Inc.
Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at firstname.lastname@example.org.