As shown in Fig 4A, incubation of Cy5-ubiquitin with E1 and ATP results in the formation of a high molecular weight adduct that is sensitive to treatment with a reducing agent indicating that the E1-thioester has been formed (lanes 3 and 4).
We identified both unconjugated Flag-Smt3 proteins as well as several higher-molecular-weight adducts.
Both lower- and higher-molecular-weight adducts of SUMO2 were purified with a preference for higher-molecular-weight chains (5-mer, 6-mer or 7-mer).
In the presence of glucosamine, the low-molecular-weight aldehydic adducts in activated extracts disappeared whereas the intensity of high-molecular-weight adducts decreased, indicating that glucosamine prevented oxidation and/or fragmentation of chondrocyte matrix components.
It is likely that when MDA or low-molecular-weight adducts is present, the level of oxidized proteins increases, and proteolytic activity could be accelerated, leading to muscle atrophy.
On immunoblot analysis of the effect of glucosamine, we identified activation-dependent low-molecular-weight MDA adduct in chondrocyte matrix extracts; the intensity of higher-molecular-weight aldehydic adducts increased in activated chondrocyte extracts as compared with extracts from control chondrocyte matrix.
The resulting MS/MS2 data were searched against an in-house generated authentic standard library that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments, includingtheir associated MS/MS spectra for all molecules in the library.
We additionally observed higher molecular weight AMP*-ligase adducts for Rnl2tr, Rnl2tr+MBP, R55K and K225R.
Metabolites were identified by automated comparison of the ion features in the experimental samples to an in-house reference library composed of more than 3,000 authentic chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as their associated MS/MS2 spectra.
Increased reactivity and appearance of low-molecular-weight aldehyde-protein adducts suggest activation-dependent aldehydic protein oxidation and protein fragmentation.
