Yulin Qi

On the isobaric space of 25-hydroxyvitamin D in human serum: potential for interferences in liquid chromatography/tandem mass spectrometry, systematic errors and accuracy issues

RATIONALE Isobaric interferences in human serum can potentially influence the measured concentration levels of 25-hydroxyvitamin D [25(OH)D], when low resolving power liquid chromatography/tandem mass spectrometry (LC/MS/MS) instruments and non-specific MS/MS product ions are employed for analysis. In this study, we provide a detailed characterization of these interferences and a technical solution to reduce the associated systematic errors. METHODS Detailed electrospray ionization Fourier transform ion cyclotron resonance (FTICR) high-resolution mass spectrometry (HRMS) experiments were used to characterize co-extracted isobaric components of 25(OH)D from human serum. Differential ion mobility spectrometry (DMS), as a gas-phase ion filter, was implemented on a triple quadrupole mass spectrometer for separation of the isobars. RESULTS HRMS revealed the presence of multiple isobaric compounds in extracts of human serum for different sample preparation methods. Several of these isobars had the potential to increase the peak areas measured for 25(OH)D on low-resolution MS instruments. A major isobaric component was identified as pentaerythritol oleate, a technical lubricant, which was probably an artifact from the analytical instrumentation. DMS was able to remove several of these isobars prior to MS/MS, when implemented on the low-resolution triple quadrupole mass spectrometer. CONCLUSIONS It was shown in this proof-of-concept study that DMS-MS has the potential to significantly decrease systematic errors, and thus improve accuracy of vitamin D measurements using LC/MS/MS.

Seven new microcystin variants discovered from a native Microcystis aeruginosa strain – unambiguous assignment of product ions by tandem mass spectrometry

RATIONALE High-resolution mass spectrometry was applied to the study of a Microcystis aeruginosa strain previously reported as a [D-Leu(1)]MC-LR producer. Detailed analysis revealed new microcystin (MC) variants produced from the strain, and seven of these were previously unreported variants. This work shows the importance of mass accuracy for the identification of unknown MCs. METHODS The M. aeruginosa strain was isolated from a bloom sample collected from Argentina and acclimated to lab conditions. The MC variants in the strain were separated by UV/Vis detection-guided high-performance liquid chromatography, and their structures were unambiguous determined by tandem mass spectrometry (MS/MS). RESULTS A simple strategy was developed for quickly locating the low-abundance MC precursors from complex samples. MS/MS anlysis revealed ten MC variants produced from the strain, of which seven have never been reported before. CONCLUSIONS This work shows the interference of isobarics and isomers in the study of unknown MCs, and, therefore, high mass accuracy is important to avoid false assignments. Moreover, the peak list provided here (30-50 fragments unambiguously assigned for ten MCs) can be used as a reference for the discovery of MCs from environmental samples.

Electron-based fragmentation methods in mass spectrometry: An overview

Tandem mass spectrometry (MS/MS) provides detailed information for structural characterization of biomolecules. The combination of electron capture dissociation (ECD) techniques with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) often provides unique ion-electron reactions and fragmentation channels in MS/MS. ECD is often a complimentary, sometimes even a superior tool to conventional MS/MS techniques. This article is aimed at providing a short overview of ECD-based fragmentation techniques (ExD) and optimization of ECD experiments for FTICR mass analyzers. Most importantly, it is meant to pique the interest of potential users for this exciting research field.

Two-dimensional mass defect matrix plots for mapping genealogical links in mixtures of lignin depolymerisation products

AbstractLignin is the second most abundant natural biopolymer, and lignin wastes are therefore potentially significant sources for renewable chemicals such as fuel compounds, as alternatives to fossil fuels. Waste valorisation of lignin is currently limited to a few applications such as in the pulp industry, however, because of the lack of effective extraction and characterisation methods for the chemically highly complex mixtures after decomposition. Here, we have implemented high resolution mass spectrometry and developed two-dimensional mass defect matrix plots as a data visualisation tool, similar to the Kendrick mass defect plots implemented in fields such as petroleomics. These 2D matrix plots greatly simplified the highly convoluted lignin mass spectral data acquired from Fourier transform ion cyclotron resonance (FTICR)–mass spectrometry, and the derived metrics provided confident peak assignments and strongly improved structural mapping of lignin decomposition product series from the various linkages within the lignin polymer after electrochemical decomposition. Graphical Abstract2D mass defect matrix plot for a lignin sample after decomposition

Detailed Study of Cyanobacterial Microcystins Using High Performance Tandem Mass Spectrometry

AbstractMicrocystins (MC) are a large group of toxic cyclic peptides, produced by cyanobacteria in eutrophic water systems. Identification of MC variants mostly relies on liquid chromatography (LC) combined with collision-induced dissociation (CID) mass spectrometry. Deviations from the essential amino acid complement are a common feature of these natural products, which makes the CID analysis more difficult and not always successful. Here, both CID and electron capture dissociation (ECD) were applied in combination with ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry to study a cyanobacteria strain isolated from the Salto Grande Reservoir in Sao Paulo State, Brazil, without prior LC separation. CID was shown to be an effective dissociation technique for quickly identifying the MC variants, even those that have previously been difficult to characterize by CID. Moreover, ECD provided even more detailed and complementary information, which enabled us to precisely locate metal binding sites of MCs for the first time. This additional information will be important for environmental chemists to study MC accumulation and production in ecosystems. Figureᅟ

Determining the Binding Sites of β-Cyclodextrin and Peptides by Electron-Capture Dissociation High Resolution Tandem Mass Spectrometry

AbstractCyclodextrins (CDs) are a group of cyclic oligosaccharides, which readily form inclusion complexes with hydrophobic compounds to increase bioavailability, thus making CDs ideal drug excipients. Recent studies have also shown that CDs exhibit a wide range of protective effects, preventing proteins from aggregation, degradation, and folding. These effects strongly depend on the binding sites on the protein surface. CDs only exhibit weak interactions with amino acids, however; conventional analytical techniques therefore usually fail to reveal the exact location of the binding sites. Moreover, some studies even suggest that CD inclusion complexes are merely electrostatic adducts. Here, electron capture dissociation (ECD) was applied in this proof-of-concept study to examine the exact nature of the CD/peptide complexes, and CD binding sites were unambiguously located for the first time via Fourier-transform ion cyclotron resonance (FTICR) tandem mass spectrometry. Graphical Abstractᅟ

Decay Mechanisms of Protonated 4-Quinolone Antibiotics After Electrospray Ionization and Ion Activation

AbstractThis study presents a detailed experimental investigation of charge isomers of protonated 4-quinolone antibiotics molecules formed during electrospray ionization (ESI) with proposed dissociation mechanisms after collisional activation. Piperazinyl quinolones have been previously shown to exhibit erratic behavior during tandem MS analyses of biological samples, which originated from varying ratios of two isomeric variants formed during ESI. Here, a combination of ESI-collision-induced dissociation (CID), differential ion mobility spectrometry (DMS), high resolution MS, and density functional theory (DFT) was used to investigate the underlying mechanisms of isomer formation and their individual dissociation behaviors. The study focused on ciprofloxacin; major findings were confirmed using structurally related 4-quinolones. DFT calculations showed a reversal of basicity for piperazinyl quinolones between liquid and gas phase. We provide an experimental comparison and theoretical treatment of factors influencing the formation ratio of the charge isomers during ESI, including solvent pH, protic/aprotic nature of solvent, and structural effects such as pKa and proton affinity. The actual dissociation mechanisms of the isomers of the protonated molecules were studied by separating the individual isomers via DMS-MS, which allowed type-specific CID spectra to be recorded. Both primary CID reactions of the two charge isomers originated from the same carboxyl group by charge-remote (CO2 loss) and charge-mediated (H2O loss) fragmentation of the piperazinyl quinolones, depending on whether the proton resides on the more basic keto or the piperazinyl group, followed by a number of secondary dissociation reactions. The proposed mechanisms were supported by calculated energies of precursors, transition states, and products for competing pathways. Graphical Abstractᅟ

Shedding light on the structures of lignin compounds: photo-oxidation under artificial UV light and characterization by high resolution mass spectrometry

AbstractLignin is the second most abundant natural polymer and a promising alternative energy source for conventional fossil fuels. In this study, we investigated transformations of lignin compounds under artificial UV light conditions at the molecular level. Such light-induced changes of composition profiles in nature after sun exposure have been studied for crude oil in the petroleomics field. We applied a similar high resolution mass spectrometry experimental strategy to lignin and demonstrated various data processing methods to reveal the characteristic differences between the extremely complex data sets of two sample sets, one native control before and one sample after photo-irradiation, using Fourier transform ion cyclotron resonance-mass spectrometry. Graphical abstractKendrick mass defect versus nominal Kendrick mass for mass spectra of a control and UV-oxidized lignin sample

Letter: β-Cyclodextrin affects the formation of isomerization products during peptide deamidation

Cyclodextrins (CDs) are a group of nontoxic oligosaccharides that are widely used as drug excipients and protein stabilizers. CDs have also been found to reduce the neurotoxicity and fibrillation of amyloid beta (Aβ), the major component of the amyloid plaques found in the brain of patients suffering from Alzheimers disease. The formation of these plaques was found to be enhanced by the presence of iso-aspartic acid (isoAsp) residues in the Aβ peptide, which can be formed by deamidation from asparagine (Asn). To explore further the influence of CDs on Aβ, we investigated three Asn-containing peptides, including Aβ25–35, by electrospray ionization, electron capture dissociation, and Fourier-transform ion cyclotron resonance mass spectrometry to explore details of the deamidation process in the presence and absence of peptide/CD adducts. The results showed that CDs reduced the formation of the isomerization product isoAsp during peptide deamidation. This finding might help to better understand the role of CDs during the protein-aggregation process.

Aggression behaviour induced by oral administration of the Janus-kinase inhibitor tofacitinib, but not oclacitinib, under stressful conditions

Janus kinase (JAK) inhibitors have recently been developed for allergic diseases. We focused on the 2 different JAK inhibitors, tofacitinib (selective for JAK3) and oclacitinib (selective for JAK1 and 2), to clarify the mechanism of anti-inflammatory and anti-itching potency of these drugs. In the process of detecting anti-itching potency, we observed that tofacitinib treated mice showed aggression behaviour. The objective of the study reported here was to investigate the aggressive behaviour induced by tofacitinib by using a mouse model of allergic dermatitis and the resident-intruder test. For the allergic dermatitis model, female BALB/c mice were sensitised and challenged topically with toluene-2,4-diisocyanate (TDI). Vehicle, tofacitinib or oclacitinib, was administered orally 30 min before TDI challenge. Scratching, aggression and standing behaviours were monitored in the 60 min period immediately following challenge of TDI. Another group of male BALB/c mice treated with vehicle, tofacitinib or oclacitinib was evaluated in the resident-intruder test and brains were obtained to determine blood brain barrier penetration. In the allergic dermatitis model, a significant increase in aggression and standing behaviour was only obvious in the tofacitinib treatment group. There was no effect in non-sensitised mice, but similar aggression was also induced by tofacitinib in male resident-intruder test. Penetration of blood-brain barrier was observed both in tofacitinib and oclacitinib treated mice. These results suggest that aggression was induced by tofacitinib under some kind of stressful environment. This study indicates a possible role of the JAK-STAT pathway in modulation of aggression behaviour.