Motif analysis was done using all SUMO remnant peptides in a SUMO remnant motif antibody PTMScan® study. Cellular WaLP peptides from HeLa cells or treated with heat shock, were immunoprecipitated with SUMO remnant motif antibody K-ε-GG and analyzed by OrbiTrap MS. The study gave 841 non-redundant sites. The Motif logo reflects the relative prevalence of an amino acid in each position relative to the 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.
|Product Includes||Volume (with Count)|
|PTMScan® Branch Motif (K-ε-GG) Immunoaffinity Beads||10 x 80 µl|
|PTMScan® IAP Buffer (10X) 9993||10 x 600 µl|
|PTMScan® Wild Type Alpha-Lytic Protease (WaLP) 33036||4 x 400 µg|
|PTMScan® Limited Use License||1 x 1 ml|
Important: Wild type alpha-lytic protease (WaLP) is a serine endopeptidase that cleaves at the carboxyl terminal side of amino acids alanine, serine, threonine, and valine. Please check that the predicted SUMO sequence of your model organism contains AGG, SGG, TGG or VGG at the c-terminus to ensure reactivity.
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 is 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/services/index.html
Small ubiquitin-related modifier 1, 2, and 3 (SUMO-1, -2, and -3) are members of the ubiquitin-like protein family (1). The covalent attachment of the SUMO-1, -2, or -3 (SUMOylation) to target proteins is analogous to ubiquitination. This post-translational modification is a reversible, multi-step process that is initiated by cleaving a precursor protein to a mature protein. Mature SUMO-1, -2, or -3 is then linked to the activating enzyme E1 and conjugated to E2. In conjunction with E3, SUMO-1, -2, or -3 is ligated to the target protein (2). Ubiquitin and the individual SUMO family members are all targeted to different proteins with diverse biological functions. Ubiquitin predominantly regulates degradation of its target (1). In contrast, SUMO-1 is conjugated to RanGAP, PML, p53, and IκB-α to regulate nuclear trafficking, formation of subnuclear structures, regulation of transcriptional activity, and protein stability (3-7). SUMO-2/-3 forms poly-(SUMO) chains, is conjugated to topoisomerase II and APP, regulates chromosomal segregation and cellular responses to environmental stress, and plays a role in the progression of Alzheimer disease (8-11).
In this assay, PTMScan® Branch Motif (K-ε-GG) Immunoaffinity Beads, a proprietary branch (“K-ε-GG”) antibody with specificity for a di-glycine tag that is the remnant of SUMO left on protein substrates after WaLP digestion, is used to enrich sumoylated peptides from WaLP-digested cell samples. This enrichment is followed by LC-MS/MS analysis for quantitative profiles of hundreds to over a thousand non-redundant sumoylated sequences.