|Product Includes||Volume (with Count)|
|PTMScan® IAP Buffer (10X) 9993||10 x 600 µl|
|PTMScan® Malonyl-Lysine [Mal-K] Immunoaffinity Beads||10 x 80 µl|
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.
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 malonyltransferase, nonenzymatic protein malonylation is likely driven by the concentration of Malonyl-CoA and intracellular pH and is subject to metabolic fluctuations (7). Malonylation is especially prevalent among mitochondrial metabolic proteins. In type II diabetes mouse models, notably elevated malonylation can be detected mainly, but not exclusively, on proteins of glucose and fatty acid metabolism (8). Yeast histone H3K56 malonylation suggests poor DNA binding efficiency and may lead to reduced cell viability (9).
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