Antioxidants Discovery for Differentiation of Monofloral Stingless Bee Honeys Using Ambient Mass Spectrometry and Metabolomics Approaches.

Stingless bee honey (SBH) is a natural, sweet product produced by stingless bees (Meliponini tribe) that has been used as a traditional medicine to treat various illnesses. It has been shown that SBH has high nutritional value and health-promoting properties due to the presence of plant bioactive compounds from different botanical flora of the foraged nectar. In this study, the antioxidant activities of seven monofloral honeys from acacia, agarwood, coconut, dwarf mountain pine (DMP), Mexican creeper (MC), rubber, and starfruit botanical origins were investigated. The antioxidant properties of SBH studied had a range from 19.7 to 31.4 mM TE/mg for DPPH assays, 16.1 to 29.9 mM TE/mg for ABTS assays, 69.0 to 167.6 mM TE/mg for ORAC assays, and 45.5 to 89.3 mM Fe2+/mg for FRAP assays. Acacia honey showed the highest level of antioxidant properties. The models built from mass spectral fingerprints from direct ambient mass spectrometry showed distinct clusters of SBH by botanical origin and correlated with the antioxidant properties. An untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics approach was undertaken to identify the antioxidant compounds that could explain the unique antioxidant and compositional profiles of the monofloral SBH by its botanical origin. The antioxidants that were identified predominantly consisted of alkaloids and flavonoids. Flavonoid derivatives, which are potent antioxidants, were found to be key markers of acacia honey. This work provides the fundamental basis for the identification of potential antioxidant markers in SBH associated with the botanical origin of the foraged nectar.

A novel screening method for free non-standard amino acids in human plasma samples using AccQ·Tag reagents and LC-MS/MS.

There are at least 500 naturally occurring amino acids, of which only 20 standard proteinogenic amino acids are used universally across all organisms in the synthesis of peptides and proteins. Non-standard amino acids can be incorporated into proteins or are intermediates and products of metabolic pathways. While the analysis of standard amino acids is well-defined, the analysis of non-standard amino acids can be challenging due to the wide range of physicochemical properties, and the lack of both reference standards and information in curated databases to aid compound identification. It has been shown that the use of an AccQ·Tag™ derivatization kit along with LC-MS/MS is an attractive option for the analysis of free standard amino acids in complex samples because it is fast, sensitive, reproducible, and selective. It has been demonstrated that the most abundant quantitative transition for MS/MS analysis of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acids corresponds to the fragmentation of the molecule at the 6-aminoquinoline carbonyl group producing a common m/z 171 fragment ion and occurs at similar mass spectrometry collision energy and cone voltages. In this study, the unique properties of AQC derivatized amino acids producing high intensity common fragment ions, along with chromatographic separation of amino acids under generic chromatography conditions, were used to develop a novel screening method for the detection of trace levels of non-standard amino acids in complex matrices. Structural elucidation was carried out by comparing the MS/MS fragment ion mass spectra generated with in silico predicted fragmentation spectra to enable a putative identification, which was confirmed using an appropriate analytical standard. This workflow was applied to screen human plasma samples for bioactive thiol-group modified cysteine amino acids and S-allylmercaptocysteine (SAMC), S-allylcysteine sulfoxide (SACS or alliin) and S-propenylcysteine (S1PC) are reported for the first time to be present in human plasma samples after the administration of garlic supplements.

Targeted and Nontargeted Metabolomics of Amino Acids and Bioactive Metabolites in Probiotic-Fermented Unhopped Beers Using Liquid Chromatography High-Resolution Mass Spectrometry.

Beer is one of the most popular beverages in the world. The increased popularity of craft beers has led to the development of unique beers that are alcohol-free, gluten-free, low calorie, or with functional properties through fermentation with probiotic microorganisms. In this study, functional unhopped beers were evaluated by utilizing probiotics (Lacticaseibacillus paracasei Lpc-37 and ibSium Saccharomyces cerevisiae CNCM I-3856) as starter cultures. The metabolites produced by probiotics were investigated using a nontargeted metabolomics approach and identified against metabolomics databases (Kyoto Encyclopedia of Genes and Genomes (KEGG), Human Metabolome Database (HMDB), Yeast Metabolome Database (YMDB), METLIN tandem mass spectrometry (MS/MS)). Derivatives of branched-chain (leucine) and aromatic amino acids (phenylalanine, tryptophan, and tyrosine) were enriched (one-way analysis of variance (ANOVA) p < 0.05) in probiotic-fermented unhopped beers, especially tryptophan metabolites. In addition, the synergistic effects of yeast-lactic acid bacteria (LAB) interactions led to further enrichment of higher acids such as (S)-(-)-2-hydroxyisocaproic acid, phenyllactic acid, hydroxyphenyllactic acid, and indolelactic acid. The potential pathways for the formation of novel bioactive tryptophan metabolites (indole and indoleacrylic acid) by LAB were elucidated. Altogether, probiotic LAB-fermented unhopped beer showed the highest antioxidant capacity and total phenolic content. This work provides the basis for the discovery of bioactive metabolites in probiotic-fermented foods.

Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly.

Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this data set reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome is likely to represent a core cell adhesion machinery, centred around four axes comprising ILK-PINCH-kindlin, FAK-paxillin, talin-vinculin and α-actinin-zyxin-VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community.

Isolation of integrin-based adhesion complexes.

The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter-receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry.

Microtubule-dependent modulation of adhesion complex composition.

The microtubule network regulates the turnover of integrin-containing adhesion complexes to stimulate cell migration. Disruption of the microtubule network results in an enlargement of adhesion complex size due to increased RhoA-stimulated actomyosin contractility, and inhibition of adhesion complex turnover; however, the microtubule-dependent changes in adhesion complex composition have not been studied in a global, unbiased manner. Here we used label-free quantitative mass spectrometry-based proteomics to determine adhesion complex changes that occur upon microtubule disruption with nocodazole. Nocodazole-treated cells displayed an increased abundance of the majority of known adhesion complex components, but no change in the levels of the fibronectin-binding α5ß1 integrin. Immunofluorescence analyses confirmed these findings, but revealed a change in localisation of adhesion complex components. Specifically, in untreated cells, α5-integrin co-localised with vinculin at peripherally located focal adhesions and with tensin at centrally located fibrillar adhesions. In nocodazole-treated cells, however, α5-integrin was found in both peripherally located and centrally located adhesion complexes that contained both vinculin and tensin, suggesting a switch in the maturation state of adhesion complexes to favour focal adhesions. Moreover, the switch to focal adhesions was confirmed to be force-dependent as inhibition of cell contractility with the Rho-associated protein kinase inhibitor, Y-27632, prevented the nocodazole-induced conversion. These results highlight a complex interplay between the microtubule cytoskeleton, adhesion complex maturation state and intracellular contractile force, and provide a resource for future adhesion signaling studies. The proteomics data have been deposited in the ProteomeXchange with identifier PXD001183.