PTMScan® Propionyl-Lysine [Prop-K] Kit #17848
Additional Information
This product is intended for peptide enrichment and mass spectrometry analysis. To learn more about our Proteomics Kits and Services please answer a few questions for our Proteomics group.
Product Information
Product Usage Information
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.
Storage
Antibody beads supplied in IAP buffer containing 50% glycerol. Store at -20°C. Do not aliquot the antibody.
Protocol
Product Description
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/services/index.html.
Background
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).
Protein propionyl and butyryl transferase activity has been reported for p300 and CREB-binding protein, two acetyltransferases that can autoacylate as well as target histone proteins and p53 in vitro. Sirt1 (Sir2 in yeast) has been shown to have depropionylase activity and may be a major eukaryotic depropionylase (6,7). In the cytosol, acetyl-CoA carboxylase (ACC) converts acetyl-CoA to Malonyl-CoA and the reverse reaction is catalyzed by Malonyl-CoA decarboxylase (MCD), but in the mitochondria, propionyl-CoA carboxylase takes the role of ACC. Both MCD and ACC are regulated by AMPK, glucose levels, and insulin, underscoring their importance in intermediary metabolism (8).
Protein propionyl and butyryl transferase activity has been reported for p300 and CREB-binding protein, two acetyltransferases that can autoacylate as well as target histone proteins and p53 in vitro. Sirt1 (Sir2 in yeast) has been shown to have depropionylase activity and may be a major eukaryotic depropionylase (6,7). In the cytosol, acetyl-CoA carboxylase (ACC) converts acetyl-CoA to Malonyl-CoA and the reverse reaction is catalyzed by Malonyl-CoA decarboxylase (MCD), but in the mitochondria, propionyl-CoA carboxylase takes the role of ACC. Both MCD and ACC are regulated by AMPK, glucose levels, and insulin, underscoring their importance in intermediary metabolism (8).
- Liu, Z. et al. (2014) Nucleic Acids Res 42, D531-6.
- Lee, S. (2013) Toxicol Res 29, 81-6.
- Lin, H. et al. (2012) ACS Chem Biol 7, 947-60.
- Zhang, Z. et al. (2011) Nat Chem Biol 7, 58-63.
- Du, J. et al. (2011) Science 334, 806-9.
- Chen, Y. et al. (2007) Mol Cell Proteomics 6, 812-9.
- Cheng, Z. et al. (2009) Mol Cell Proteomics 8, 45-52.
- Newman, J.C. et al. (2012) J Biol Chem 287, 42436-43.
限制使用
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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
AcetylScan is a registered trademark of Cell Signaling Technology, Inc.
MethylScan is a registered trademark of Cell Signaling Technology, Inc.
PhosphoScan is a registered trademark of Cell Signaling Technology, Inc.
UbiScan is a registered trademark of Cell Signaling Technology, Inc.
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