Background Nonalcoholic fatty liver disease (NAFLD) is a common metabolic disease that affects 20–30% of individuals worldwide. Liver puncture remains the gold standard for the diagnosis of liver diseases despite limitations regarding invasive nature and sample variability. It is of great clinical significance to find noninvasive biomarkers to detect and predict NAFLD. Objective The aims of this study were to identify potential serum markers in individuals with early-stage NAFLD and to advance the mechanistic understanding of this disease using a high-throughput mass spectrometry-based untargeted metabolomics approach. Methods One hundred and twelve patients with early-stage NAFLD aged 18–55 were recruited according to the guidelines. The control group included 112 healthy participants. The demographic, anthropometric, clinical and laboratory data of all participants were systematically collected. Serum samples were obtained after an overnight fast. The comprehensive serum metabolomic analysis was performed by ultra-performance liquid chromatography-Orbitrap mass spectrometry. The resultant data was processed by Compound Discover and SIMCA-P software to validate the potential biomarkers. Significantly altered metabolites were evaluated by variable importance in projection value (VIP > 1) and ANOVA (p < 0.01). Pathway analysis was performed using MetaboAnalyst 4.0. Results The liver function test of early NAFLD patients showed no statistical differences to control group (p > 0.05). However, obvious differences in blood lipids were observed between subjects with NAFLD and controls (p < 0.001). In total, 55 metabolites showed significant changes in experimental group were identified. The area under curve (AUC) values deduced by receiver operating curve (ROC) analysis indicated that these newly identified biomarkers have high predictability and reliability. Of these, 15 metabolites with AUC greater than 0.9 were of great diagnostic value in early NAFLD patients. Conclusion In this study, a total of 15 serum metabolites were found to strongly associate with early NAFLD. These biomarkers may have great clinical significance in the early diagnosis of NAFLD, as well as to follow response to therapeutic interventions.

It remains particularly difficult for gaining unambiguous information on anomer, linkage, and position isomers of oligosaccharides using conventional mass spectrometry (MS) methods. In our laboratory, an ion mobility (IM) shift strategy was employed to improve confidence in the identification of structurally closely related disaccharide and monosaccharide isomers using IMMS. Higher separation between structural isomers was achieved using 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization in comparison with phenylhydrazine (PHN) derivatization. Furthermore, the combination of pre-IM fragmentation of PMP derivatives provided sufficient resolution to separate the isomers not resolved in the IMMS. To chart the structural variation observed in IMMS, the collision cross sections (CCSs) for the corresponding ions were measured. We analyzed nine disaccharide and three monosaccharide isomers that differ in composition, linkages, or configuration. Our data show that coexisting carbohydrate isomers can be identified by the PMP labeling technique in conjunction with ion-mobility separation and tandem mass spectrometry. The practical application of this rapid and effective method that requires only small amounts of sample is demonstrated by the successful analysis of water-soluble ginseng extract. This demonstrated the potential of this method to measure a variety of heterogeneous sample mixtures, which may have an important impact on the field of glycomics.

Cu, Zn-superoxide dismutase (SOD1) is a homodimeric enzyme of approximately 32 kDa. Each monomer contains one Cu(2+) and one Zn(2+) ion, which play catalytic and structural roles in the enzyme. Dimer formation is also essential to its functionality. The spatial structure of this metalloenzyme is also closely related to its bioactivities. Here we investigate the structural and conformational changes of SOD1 in the gas phase by electrospray ionization mass spectrometry (ESI-MS) and ion-mobility (IM) separation combined with tandem mass spectrometry (MS/MS). First, the composition and forms of SOD1 were analyzed by ESI-MS. The dimer, monomer, and apomonomer were observed under different solvent conditions. The dimer was found to be stable, and could retain its native structure in neutral buffer. Ion-mobility separation combined with MS/MS was used to reveal the conformational changes and dissociation process of SOD1 when it was activated in the gas phase. Three different dimeric and two monomeric conformers were observed; three unfolding and dissociation pathways were also identified. The results from this study demonstrate that IM-MS/MS could be used to obtain spatial structural information on SOD1 and that the technique could therefore be employed to investigate the conformational changes in mutant SOD1, which is related to amyotrophic lateral sclerosis and other neurodegenerative disorders.

Misfolding and aggregation of Cu, Zn superoxide dismutase (SOD1) is implicated in the etiology of amyotrophic lateral sclerosis (ALS). The use of small molecules may stabilize the spatial structure of SOD1 dimer, thus, preventing its dissociation and aggregation. In this study, "native" mass spectrometry (MS) was used to study the noncovalent interactions between SOD1 and flavonoid compounds. MS experiments were performed on a quadruple time-of-flight (Q-ToF) mass spectrometer with an electrospray ionization (ESI) source and T-wave ion mobility. ESI-MS was used to detect the SOD1-flavonoid complexes and compare their relative binding strengths. The complement of ion mobility separation allowed comparison in the binding affinities between flavonoid isomers and provided information on the conformational changes. Molecular docking together with molecular dynamics simulations and MM/PBSA methods were applied to gain insights into the binding modes and free energies of SOD1-flavonoid complexes at the molecule level. Among all the flavonoids investigated, flavonoid glycosides preferentially bind to SOD1 than their aglycone counterparts. Naringin, one of the compounds that has the strongest binding affinity to SOD1, was subjected to further characterization. Experiment results show that the binding of naringin can stabilize SOD1 dimer and inhibit the aggregation of SOD1. Molecular simulation results suggest that naringin could reduce the dissociation of SOD1 dimers through direct interaction with the dimer interface. This developed analytical strategy could also be applied to study the interactions between SOD1 and other drug-like molecules, which may have the effect to reduce the aggregation.

We investigate the influence of three volatile alkylammonium acetate buffers on binding affinities for protein-ligand interactions determined by native electrospray ionization-mass spectrometry (ESI-MS). Four different types of proteins were chosen for this study. A charge-reduction effect was observed for all the cases studied, in comparison to the ions formed in ammonium acetate solution. When increasing the collision energy, the complexes of trypsin and the ligand were found to be more stable when sprayed from alkylammonium acetate buffers than from ammonium acetate. The determined dissociation constant (K) also exhibited a drop (up to 40%) when ammonium acetate was replaced by alkylammonium acetate buffers for the case of lysozyme and the ligand. The prospective uses of these ammonium acetate analogs in native ESI-MS are discussed in this paper as well. Graphical Abstract ᅟ.

Multidrug resistance (MDR) is one of the major obstacles for clinical effective chemotherapy. In this study, the effects and possible mechanisms of aloe-emodin (AE) were investigated on reversing the adriamycin (ADR)-induced resistance of MCF-7/ADR cells. AE could significantly reverse the ADR resistance in MCF-7/ADR cells. The combination of AE (20 μM) and ADR had no effect on the P-glycoprotein (P-gp) level, but notably promoted the accumulation of ADR in drug-resistant cells. The efflux function of P-gp required ATP, but AE reduced the intracellular ATP level. AE played a reversal role might through inhibiting the efflux function of P-gp. The research result of energy metabolism pathways indicated that combination of AE and ADR could inhibit glycolysis, tricarboxylic acid (TCA) cycle, glutamine metabolism, and related amino acid synthesis pathways. Moreover, we found AE not only reversed ADR-induced resistant but also induced autophagy as a defense mechanism. In addition, the combination of AE and ADR arrested G2/M cell cycle and induced apoptosis through DNA damage, ROS generation, caspase-3 activation. Our study indicated that AE could be a potential reversal agent to resensitize ADR resistant in tumor chemotherapy and inhibiting autophagy might be an effective strategy to further enhance the reversal activity of AE.

The aggregation of Cu,Zn-superoxide dismutase (SOD1) plays an important role in the etiology of amyotrophic lateral sclerosis (ALS). For the disruption of ALS progression, discovering new drugs or compounds that can prevent SOD1 aggregation is important. In this study, ESI-MS was used to investigate the interaction of catechins and SOD1. The noncovalent complex of catechins that interact with SOD1 was found and retained in the gas phase under native ESI-MS condition. The conformation changes of SOD1 after binding with catechins were also explored via traveling wave ion mobility (IM) spectrometry. Epigallocatechin gallate (EGCG) can stabilize SOD1 conformation against unfolding in three catechins. To further evaluate the efficacy of EGCG, we monitored the fluorescence changes of dimer E,E,-SOD1(apo-SOD1, E:empty) with and without ligands under denaturation conditions, and found that EGCG can inhibit apo-SOD1 aggregation. In addition, the circular dichroism spectra of the samples showed that EGCG can decrease the β-sheet content of SOD1, which can produce aggregates. These results indicated that orthogonal separation dimension in the gas-phase IM coupled with ESI-MS (ESI-IM-MS) can potentially provide insight into the interaction between SOD1 and small molecules. The advantage is that it dramatically decreases the analysis time. Meantime, optical spectroscopy techniques can be used to confirm ESI-IM-MS results. Graphical Abstract ᅟ.

To inhibit the abnormal aggregation of Cu, Zn-superoxide dismutase (SOD1) is regarded as a potential therapeutic strategy of SOD1-linked amyotrophic lateral sclerosis (ALS). Herein the interactions between SOD1 and four stilbene-based polyphenols, namely, resveratrol, oxyresveratrol, polydatin, and 2,3,4′,5-tetrahydroxystilbene-2-O-β-d-glycoside (THSG), were investigated using electrospray ionization mass spectrometry (ESI-MS) combined with ion mobility (IM) spectrometry. The addition of tandem MS to the study of SOD1–ligand complexes provides further insight into their gas-phase stability. Monitoring the unfolding of SOD1–ligand complexes using IM-MS allows observation of subtle changes in the protein stability upon ligand binding. From the MS/MS and IM-MS measurements, polydatin and THSG were highlighted as the strongest bound compounds in the gas phase, and both of them appear to provide a stabilizing effect on the SOD1 dimer conformation. In addition, the data of fluorescence assays clearly show the ability of the ligands to inhibit apoSOD1 from aggregation, and polydatin was found to have the strongest inhibitory effect. Overall, the method described here can be an effective approach to investigate the interactions between SOD1 and other drug-like molecules.