Overview
This website contains ubiquitinated loci from Arabidopsis thaliana.
To gain insight into the Arabidopsis ubiquitinome, we used the ubiquitin COFRADIC technology (Stes et al., 2014) with some minor modifications with respect to protein preparation. This technique allows the enrichment of ubiquitinated peptides, starting from plant cell. In short, proteins were extracted via methanol/chloroform precipitation and the pellet was resolubilized in 6 M guanidinium hydrochloride. After reduction, alkylation, and desalting, the protein extracts were chemically acetylated to block all amino groups at protein N-termini and on lysine side chains. The proteins were incubated with the catalytic center of the human USP2 deubiquitinase (USP2cc) that removes enzymatically (acetylated) ubiquitin. The freed amino groups were subsequently chemically modified with a handle that allows specific isolation of ubiquitinated peptides during COFRADIC separations. Finally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the isolated fractions allowed identification of peptide and ubiquitin sites.
To gain insight into the Arabidopsis ubiquitinome, we used the ubiquitin COFRADIC technology (Stes et al., 2014) with some minor modifications with respect to protein preparation. This technique allows the enrichment of ubiquitinated peptides, starting from plant cell. In short, proteins were extracted via methanol/chloroform precipitation and the pellet was resolubilized in 6 M guanidinium hydrochloride. After reduction, alkylation, and desalting, the protein extracts were chemically acetylated to block all amino groups at protein N-termini and on lysine side chains. The proteins were incubated with the catalytic center of the human USP2 deubiquitinase (USP2cc) that removes enzymatically (acetylated) ubiquitin. The freed amino groups were subsequently chemically modified with a handle that allows specific isolation of ubiquitinated peptides during COFRADIC separations. Finally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the isolated fractions allowed identification of peptide and ubiquitin sites.
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Annotated spectra
Download the annotated spectra information in PDF format.
What is ubiquitination?
An important PTM is the conjugation of the small (8.5 kDa), highly conserved, and abundant protein ubiquitin to target proteins.
Ubiquitin has a flexible C-terminus, terminating with a glycine residue that via an isopeptidyl bond can be attached covalently with ε-amino groups of lysine residues on substrates (Heride et al., 2014).
Ubiquitin is linked to the target protein by the consecutive action of three types of proteins, collectively known as the E1-E2-E3 conjugation cascade.
Whereas E1 and E2 are involved in activation and transfer of ubiquitin, the E3 ligase confers the modification reaction specificity through its recognition of target proteins, directly or indirectly catalyzing the ligation to the ubiquitin chain (Chen and Hellmann, 2013).
Although the most commonly described sites for this ligation are lysine residues, ubiquitination is also known to occur on the side chains of other amino acids, such as cysteine, serine, and threonine (Iwai and Tokunaga, 2009;Shimizu et al., 2010;Okumoto et al., 2011). Additionally, examples of ubiquitination of the N-terminal α-amino group of proteins have been reported (Scaglione et al., 2013;Tatham et al., 2013;Vittal et al., 2015), but the incidence of this type of ubiquitination is currently unexplored in plants.
Besides regulation of the protein longevity via targeted degradation by the ubiquitin proteasome system (UPS), ubiquitination can alter protein activity, localization, and interactions (Hua and Vierstra, 2011). As ubiquitin can form linear or branched chains through linkage of ubiquitin chains to its own N-terminus or internal lysine residues, respectively, a large diversity in ubiquitination types exists, each presumably affecting protein fate in a different manner (Komander and Rape, 2012).
Although the most commonly described sites for this ligation are lysine residues, ubiquitination is also known to occur on the side chains of other amino acids, such as cysteine, serine, and threonine (Iwai and Tokunaga, 2009;Shimizu et al., 2010;Okumoto et al., 2011). Additionally, examples of ubiquitination of the N-terminal α-amino group of proteins have been reported (Scaglione et al., 2013;Tatham et al., 2013;Vittal et al., 2015), but the incidence of this type of ubiquitination is currently unexplored in plants.
Besides regulation of the protein longevity via targeted degradation by the ubiquitin proteasome system (UPS), ubiquitination can alter protein activity, localization, and interactions (Hua and Vierstra, 2011). As ubiquitin can form linear or branched chains through linkage of ubiquitin chains to its own N-terminus or internal lysine residues, respectively, a large diversity in ubiquitination types exists, each presumably affecting protein fate in a different manner (Komander and Rape, 2012).