Nutritional composition and bioactive compounds of Melipona seminigra pot-pollen from Amazonas, Brazil.

BACKGROUND: Pot-pollen is a fermented product stored by stingless bees in cerumen pots and traditionally used as food or medicine by natives in tropical regions. Knowledge of pot-pollen composition from the Amazon region is important to strengthen the breeding of native bees and consequently contribute to sustainable development in this region. The aim of the present study was to determine the chemical composition of Melipona seminigra pot-pollen from Amazonas, Brazil. RESULTS: We report the identification of 21 phenolic compounds using ultra-performance liquid chromatography- electrospray ionization-quadrupole time-of-flight-mass spectrometry. Overall, the predominant constituents of pot-pollen were protein (333.8-470.7 g kg-1 ) and dietary fibers (247.6-353.3 g kg-1 ). The predominant fatty acids were polyunsaturated (44.95-59.57% of total fatty acid content) and the samples contained all essential amino acids. Total carotenoid content ranged from 3.2 to 48.0 µg g-1 , total flavonoid content (as catechin equivalents) from 1.9 to 4.5 mg g-1 , total reducing capacity (as gallic acid equivalents) from 7.0 to 15.0 mg g-1 , ferric-reducing antioxidant power from 2.8 to 8.9 µmol g-1 and oxygen radical absorbance capacity (as Trolox equivalents) from 224.9 to 1117.0 µmol g-1 . CONCLUSIONS: This study reports for the first time a comprehensive chemical characterization of M. seminigra pot-pollen. The samples presented antioxidant activity, high values of nutrients and bioactive compounds such as polyphenols, carotenoids, insoluble dietary fibers, polyunsaturated fatty acids, and essential amino acids, comparable to other health foods. © 2021 Society of Chemical Industry.

A new label-free approach for the determination of reaction rates in oxidative footprinting experiments.

Hydroxyl radical-mediated oxidative footprinting coupled to mass spectrometric analysis is an attractive technique for protein surface mapping, conformational changes monitoring, and protein-ligand interfaces mapping in solution. In this technique, a protein is oxidized by in situ-generated hydroxy radicals and the site and rate of oxidation can be determined by proteolysis followed by mass spectrometric analysis. Changes in peptide oxidation rate can then be correlated to the changes in solvent exposure, and information about conformational changes or interaction domains can be obtained. The method relies, therefore, on the accurate measurements of peptide oxidation rate. Here, we describe a new label-free method to determine the oxidation rate of peptides that is based on the consumption of the unoxidized peptide instead of measuring the formation of oxidized peptides. The reaction rate thus obtained presents a better linearity and lower variation when compared to the traditional method. The label-free method is also simpler to implement and automation can be achieved through label-free quantitation software.

Fragmentation features of intermolecular cross-linked peptides using N-hydroxy- succinimide esters by MALDI- and ESI-MS/MS for use in structural proteomics.

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.

Traveling-wave ion mobility mass spectrometry analysis of isomeric modified peptides arising from chemical cross-linking.

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 conditions.

A ditryptophan cross-link is responsible for the covalent dimerization of human superoxide dismutase 1 during its bicarbonate-dependent peroxidase activity.

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.

Identification of cross-linked peptides by high-resolution precursor ion scan.

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.

Collision-induced dissociation of Lys-Lys intramolecular crosslinked peptides.

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/ionization-generated 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 crosslinker backbone and lysine (m/z 222) is formed. The detailed identification of fragment ions can help the development of softwares devoted to the MS/MS data analysis of crosslinked peptides.