Unlike intermolecular disulfide bonds, other protein cross-links arising from oxidative modifications cannot be reversed and are presumably more toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain poorly characterized. For instance, the antioxidant enzyme human superoxide dismutase 1 (hSod1) has been reported to undergo non-disulfide covalent dimerization and further oligomerization during its bicarbonate-dependent peroxidase activity. The dimerization was shown to be dependent on the oxidation of the single, solvent-exposed Trp(32) residue of hSod1, but the covalent dimer was not isolated nor was its structure determined. In this work, the hSod1 covalent dimer was isolated, digested with trypsin in H(2)O and H(2)(18)O, and analyzed by UV-Vis spectroscopy and mass spectrometry (MS). The results demonstrate that the covalent dimer consists of two hSod1 subunits cross-linked by a ditryptophan, which contains a bond between C3 and N1 of the respective Trp(32) residues. We further demonstrate that the cross-link cleaves under usual MS/MS conditions leading to apparently unmodified Trp(32), partially hinders proteolysis, and provides a mechanism to explain the formation of hSod1 covalent trimers and tetramers. This characterization of the covalent hSod1 dimer identifies a novel oxidative modification of protein Trp residues and provides clues for studying its occurrence in vivo.

The use of chemical crosslinking is an attractive tool that presents many advantages in the application of mass spectrometry to structural biology. The correct assignment of crosslinked peptides, however, is still a challenge because of the lack of detailed fragmentation studies on resultant species. In this work, the fragmentation patterns of intramolecular crosslinked peptides with disuccinimidyl suberate (DSS) has been devised by using a set of versatile, model peptides that resemble species found in crosslinking experiments with proteins. These peptides contain an acetylated N-terminus followed by a random sequence of residues containing two lysine residues separated by an arginine. After the crosslinking reaction, controlled trypsin digestion yields both intra-and intermolecular crosslinked peptides. In the present study we analyzed the fragmentation of matrix-assisted laser desorption/ionizationgenerated peptides crosslinked with DSS in which both lysines are found in the same peptide. Fragmentation starts in the linear moiety of the peptide, yielding regular b and y ions. Once it reaches the cyclic portion of the molecule, fragmentation was observed to occur either at the following peptide bond or at the peptide crosslinker amide bond. If the peptide crosslinker bond is cleaved, it fragments as a regular modified peptide, in which the DSS backbone remains attached to the first lysine. This fragmentation pattern resembles the fragmentation of modified peptides and may be identified by common automated search engines using DSS as a modification. If, on the other hand, fragmentation happens at the peptide bond itself, rearrangement of the last crosslinked lysine is observed and a product ion containing the M ass spectrometry for three-dimensional analysis (MS3D) has become a very attractive tool in evaluating protein structure and interactions. In MS3D, proteins are subjected to crosslinking with one of the many reagents available followed by enzymatic digestion and MS analysis [1]. In recent years, this approach has been widely used in the study of protein folding [2-6], identifying binding partners [7][8][9], monitoring conformational changes upon ligand binding [10 -12], characterizing surfaces in protein complexes [13][14][15][16][17][18][19], and as probes for solvent accessibility [20 -23].One of the key steps for a successful MS3D analysis relies on the correct assignment of crosslinked peptides. The identification of those peptides is not trivial because they are present in the sample in a low stoichiometric amount. Several approaches are currently being applied to detect these modified peptides [24 -27], with one of the most explored methodologies consisting of tagging crosslinked peptides with heavy isotopes, either by using isotopically coded crosslinkers [18, 28 -33] or tryptically digesting the protein solution in a mixture of H 2 16 O and H 2 18 O [8, 31, 34, 35]. Affinity-tagged crosslinkers have been synthesized, so that modified peptides can be enriched after reaction [28, 30, 36 -38]. Al...

BackgroundRuminants play a great role in sustainable livestock since they transform pastures, silage, and crop residues into high-quality human food (i.e. milk and beef). Animals with better ability to convert food into animal protein, measured as a trait called feed efficiency (FE), also produce less manure and greenhouse gas per kilogram of produced meat. Thus, the identification of high feed efficiency cattle is important for sustainable nutritional management. Our aim was to evaluate the potential of serum metabolites to identify FE of beef cattle before they enter the feedlot.ResultsA total of 3598 and 4210 m/z features was detected in negative and positive ionization modes via liquid chromatography-mass spectrometry. A single feature was different between high and low FE groups. Network analysis (WGCNA) yielded the detection of 19 and 20 network modules of highly correlated features in negative and positive mode respectively, and 1 module of each acquisition mode was associated with RFI (r = 0.55, P < 0.05). Pathway enrichment analysis (Mummichog) yielded the Retinol metabolism pathway associated with feed efficiency in beef cattle in our conditions.ConclusionAltogether, these findings demonstrate the existence of a serum-based metabolomic signature associated with feed efficiency in beef cattle before they enter the feedlot. We are now working to validate the use of metabolites for identification of feed efficient animals for sustainable nutritional management.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5406-2) contains supplementary material, which is available to authorized users.

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