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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Chemical cross-linking coupled to mass spectrometry analysis has become a realistic alternative to the study of proteins structure and interactions, especially when these systems are not amenable to high-resolution techniques such as protein crystallography or nuclear magnetic resonance. One of the main bottlenecks of this approach relies on the detection of cross-linked peptides, as they are usually present in substoichiometric amounts in complex samples. It was shown that one of the main fragmentation pathways of disuccinimidyl suberate (DSS) cross-linked peptides yields diagnostic ions, whose structure is composed of a rearranged lysine side chain and the spacer arm of the linker. In this report, we demonstrate the feasibility of detecting these modified peptides based on a precursor ion scan in a quadrupole time-of-flight (Q-TOF) instrument. It was shown that the fragmentation of nonmodified tryptic peptides hardly generates ions with the same nominal mass of the diagnostic ions, making the precursor ion scan very specific to N-hydroxysuccinimide (NHS)-based cross-linkers. Moreover, the experimental setup is the same as in the case of a regular cross-linking experiment, not demanding any additional experimental steps that would increase sample handling. The results obtained with protein samples allowed us to propose an algorithm that could be implemented in a software to process data from cross-linking experiments in an automated and high-throughput way.

The use of mass spectrometry coupled with chemical cross-linking of proteins has become one of the most useful tools for proteins structure and interactions studies. One of the challenges in these studies is the identification of the cross-linked peptides. The interpretation of the MS/MS data generated in cross-linking experiments using N-hydroxy succinimide esters is not trivial once a new amide bond is formed allowing new fragmentation pathways, unlike linear peptides. Intermolecular cross-linked peptides occur when two different peptides are connected by the cross-linker and they yield information on the spatial proximity of different domains (within a protein) or proteins (within a complex). In this article, we report a detailed fragmentation study of intermolecular cross-linked peptides, generated from a set of synthetic peptides, using both ESI and MALDI to generate the precursor ions. The fragmentation features observed here can be helpful in the interpretation and identification of cross-linked peptides present in cross-linking experiments and be further implemented in search engine's algorithms.

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Traveling-wave ion mobility (TWIM) coupled to mass spectrometry (MS) has emerged as a powerful tool for structural and conformational analysis of proteins and peptides, allowing the analysis of isomeric peptides (or proteins) with the same sequence but modified at different residues. This work demonstrates the use of the novel TWIM-MS technique to separate isomeric peptide ions derived from chemical cross-linking experiments, which enables the acquisition of distinct product ion spectra for each isomer, clearly indicating modification on different sites. Experiments were performed with four synthetic peptides, for which variable degrees of mobility separation were achieved. In cases of partially overlapping mobility arrival time distributions (ATDs), extracting the ATDs of fragment ions belonging to each individual isomer allowed their separation into two distinct ATDs. Accumulation over regions from the specific ATDs generates the product ion spectrum of each isomer, or a spectrum highly enriched in their fragments. The population of both modified peptide isomers was correlated with the intrinsic reactivities of different Lys residues from reactions conducted at different pH