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Characterization of Protein SUMOylation

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Creative Proteomics
New York, New York - United States
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Description & Specifications


There are a variety of cellular pathways regulated by the protein post-translational modifications with the ubiquitin-family proteins. SUMO, the Small Ubiquitin-like MOdifier, a member of the family of ubiquitin-like proteins (Ubls) and conserved from yeast to human, is covalently linked to specific target proteins at the ε-amino group of lysine residues through a process similar to the ubiquitin, catalyzed by three enzymes: an activating enzyme (E1), a conjugating enzyme (E2) and a ligating enzyme (E3).

Currently there are four SUMO isoforms identified in previous investigation. SUMO-2 and SUMO-3 share 97% sequence, and they have a 41% identity to SUMO-1; SUMO-4 is not ubiquitously expressed, only found in the immune tissues, kidney cells and pancreatic islets, but not found conjugated to any protein substrates yet. SUMO reacts with the ε-atom of certain Lys residues which results in branched peptide formation. Tryptic digestion of ubiquitylated proteins cleaves lysine/arginine residues in both the substrate and ubiquitin sequences, leaving a short KK “tag” attached to the lysine of substrate sequence. Tandem mass spectrometry can simply treat the 114 Da mass shift as a post-translational modification, which can be compatible with most search engines, like Mascot. But even after tryptic cleavage, the sequence residue of SUMO is much longer, not easily treated as just a simple PTM, because fragmentation of this tail may dominate the tandem mass spectra, reducing the ability for sequence identification using MS and MS/MS.

So for characterization of SUMOylation, the mutagenesis technique has been utilized to analyze SUMOylated structures by mass spectrometry. For example, modified SUMO-1 whose C-terminal -TGG was mutated to the ubiquitin sequence -RGG, or yeast homolog SMTP3, whose arginine residue proximal to the C-terminus, leading to shorter -TGG and -EQIGG, respectively, after tryptical cleavage. Generally the analytical approaches for identifying SUMO substrates include over expression of the SUMO isoforms with an N-terminal histidine tag for purification, as well as site-directed mutagenesis of the C-terminal end of the SUMO sequence to shorten the residual tag, to simplify SUMO spectra, and make interpretation of mass spectra and location of the SUMOylation sites easier. Coupled with various proteases and acid hydrolysis, our tech team are glad to discuss the details of clients’ projects, and provide most suitable analytical proposal.