Arbutin derivatives from the buds of Vaccinium dunalianum and their MS/MS spectrometric fragmentations

Four arbutin derivatives were isolated from the buds of Vaccinium dunalianum in which 4-hydroxyphenyl-6′-(3''-O-β-D-glucopyranosyl-4''-hydroxycinnamoyl)-O-β-D-glucopyranoside (1) was a new compound. The structure of the new compound was determined on the basis of NMR and HR-ESI-MS data. All the arbutin derivatives were subjected to the MS/MS analyses from which the MS/MS spectrometric fragmentations were summarized.     

Biological monitoring of bisphenol A with HLPC/FLD and LC/MS/MS assays

Biological monitoring is a necessary process for risk assessment of endocrine disrupting chemicals (EDCs), particularly, bisphenol A (BPA), in breast milk, because its human risks are not clear yet, and infants, who feed on breast milk, are highly susceptible for EDCs. Concerning biological monitoring of BPA, the HPLC/FLD has been widely used before the LC/MS/MS. However, there was no report, which simultaneously evaluated the two methods in real analyses. Therefore, we analyzed BPA with LC/MS/MS and HPLC/FLD in human breast milk and conducted comparison of two methods in analyzed BPA levels. After establishing optimal condition, e.g. linearity, recovery, reproducibility and free BPA system, we analyzed BPA levels in human breast milk samples (N = 100). The LOQs were similar in the two methods, i.e. 1.8 and 1.3 ng/mL for the HPLC/FLD and LC/MS/MS assays, respectively. There were strong associations between total BPA levels with the two methods (R² = 0.40, p < 0.01), however, only 11% of them were analyzed as similar levels with 15% CVs. In addition, the detection range of BPA was broader in the HPLC method than the LC/MS/MS method. However, the BPA levels in the HPLC/FLD analysis were lower than those in the LC/MS/MS analysis (p < 0.01). Thus, the differences in BPA levels between the two methods may come from mainly over-estimation with the LC/MS/MS method in low BPA samples and some of poor resolution with the HPLC/FLD in high BPA samples.     

Comparison of hydrolysis and HPLC/MS/MS procedure with ELISA assay for the determination of S-phenylmercapturic acid as a biomarker of benzene exposure in human urine

The present study compared three methods for the determination of S-phenylmercapturic acid (S-PMA), a metabolite of benzene, in human urine: a HPLC/MS/MS technique with two different sample treatments (strong and partial hydrolysis) and a commercial assay based on anti-S-PMA monoclonal antibodies with chemiluminescence detection. Biological monitoring was done on 126 volunteers and the results were compared for the three methods and also with benzene exposure levels (range <3.0–592.5 μg/m³). The correlation between environmental monitoring data and S-PMA levels in non-smokers (n = 73) was highly significant (p < 0.0001, Student's t-test) for both HPLC/MS/MS methods (r = 0.65 both for strong acidic hydrolysis of the urine and hydrolysis at pH 2) but not for the immunoassay, which overestimated the S-PMA levels by about 8 μg/g creatinine (creat.). Therefore the immunoassay is only useful as a semiquantitative screening test, but quantitative results need to be confirmed by a more accurate method like HPLC/MS/MS. The HPLC/MS/MS procedure with strong acid hydrolysis led to a recovery of S-PMA about double that using pH 2 hydrolysis, giving more accurate results. The difference between the results with the two methods makes it difficult to compare the strong acidic hydrolysis data with the ACGIH BEI value of 25 μg/g creat. since the BEI^® documentation is based on data collected in pH conditions that were not always controlled, which may underestimate the true S-PMA concentration. Besides, as levels of benzene exposure were high, smoking was not considered a confounding factor. The BEI for S-PMA in end of shift urine samples could be reconsidered when sufficient data are available from studies where the analyses are carried out in comparable conditions of hydrolysis and monitoring only non-smoking subjects.     

PPIRank - an advanced method for ranking protein-protein interations in TAP/MS data

Background

Tandem affinity purification coupled with mass-spectrometry (TAP/MS) analysis is a popular method for the identification of novel endogenous protein-protein interactions (PPIs) in large-scale. Computational analysis of TAP/MS data is a critical step, particularly for high-throughput datasets, yet it remains challenging due to the noisy nature of TAP/MS data.

Results

We investigated several major TAP/MS data analysis methods for identifying PPIs, and developed an advanced method, which incorporates an improved statistical method to filter out false positives from the negative controls. Our method is named PPIRank that stands for PPI rank ing in TAP/MS data. We compared PPIRank with several other existing methods in analyzing two pathway-specific TAP/MS PPI datasets from Drosophila.

Conclusion

Experimental results show that PPIRank is more capable than other approaches in terms of identifying known interactions collected in the BioGRID PPI database. Specifically, PPIRank is able to capture more true interactions and simultaneously less false positives in both Insulin and Hippo pathways of Drosophila Melanogaster.

     

Application of Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Rapid Identification of Neisseria Species

Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI MS) was applied to develop a proteomics-based method to detect and identify Neisseria species. Heat-inactivated clinical isolate cell suspensions of Neisseria gonorrhoeae and strains belonging to five serogroups (A, B, C, W135, and Y) of Neisseria meningitidis were subjected to on-probe protein/peptide extraction and tryptic digestion followed by AP-MALDI tandem MS (MS/MS)-based proteomic analysis. Amino acid sequences derived from three protonated peptides with m/z values of 1743.8, 1894.8, and 1946.8 were identified by AP-MALDI MS/MS and MASCOT proteome database search analysis as belonging to neisserial acyl carrier protein, neisserial-conserved hypothetical protein, and neisserial putative DNA binding protein, respectively. These three peptide masses can thus be potential biomarkers for neisserial species identification by AP-MALDI MS.     

Differential Proteome Analysis: Two-Dimensional Nano-LC/MS of E. Coli Proteome Grown on Different Carbon Sources

Different sugars provided to bacteria as single sources of carbon and energy require the induction of different metabolic enzymes, transporters, and uptake systems in order to support growth and cell survival. Using a nano–high-performance liquid chromatography/mass spectrometry (nano-HPLC/MS) system we constructed comprehensive peptide maps for Escherichia coli grown with either lactose or glucose in minimal medium. Digested bacterial samples were separated in a two-dimensional manner by combining strong cation exchange (SCX) and reversed-phased (RP) chromatography. Peptides were eluted online to an iontrap MS instrument and further analyzed by tandem MS fragmentation. Bacterial proteins originating from the differing samples were analyzed by searching the Swiss Prot Database. Data are presented that show the ability to detect several hundred different proteins significantly expressed under both conditions. Several enzymes and binding proteins related to the lactose metabolism were only identified in the sample grown with this carbon source.     

Development and validation of a high-throughput method for the quantitative analysis of d-amphetamine in rat blood using liquid chromatography/MS on a hybrid triple quadrupole-linear ion trap mass spectrometer and its application to a pharmacokinetic study

Amphetamines are a group of sympathomimetic drugs that exhibit strong central nervous system stimulant effects. d-Amphetamine ((+)-alpha-methylphenetylamine) is the parent drug in this class to which all others are structurally related. In drug discovery, d-amphetamine is extensively used either for the exploration of novel mechanisms involving the catecholaminergic system, or for the validation of new behavioural animal models. Due to this extensive use of d-amphetamine in drug research and its interest in toxicologic–forensic investigation, a specific and high-throughput method, with minimal sample preparation, is necessary for routine analysis of d-amphetamine in biological samples. We propose here a sensitive, specific and high-throughput bioanalytical method for the quantitative determination of d-amphetamine in rat blood using MS³ scan mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (LC–MS/MS/MS). Blood samples, following dilution with water, were prepared by fully automated protein precipitation with acetonitrile containing an internal standard. The chromatographic separation was achieved on a Waters XTerra C18 column (2.1 mm × 30 mm, 3.5 μm) using gradient elution at a flow rate of 1.0 mL/min over a 2 min run time. An Applied Biosystems API4000 QTRAP™ mass spectrometer equipped with turbo ion-spray ionization source was operated simultaneously in MS³ scan mode for the d-amphetamine and in multiple reaction monitoring (MRM) for the internal standard. The MS/MS/MS ion transition monitored was m/z 136.1 → 119.1 → 91.1 for the quantitation of d-amphetamine and for the internal standard (rolipram) the MS/MS ion transition monitored was m/z 276.1 → 208.2. The linear dynamic range was established over the concentration range 0.5–1000 ng/mL (r² = 0.9991). The method was rugged and sensitive with a lower limit of quantification (LLOQ) of 0.5 ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. This method was successfully applied to evaluate the pharmacokinetics of d-amphetamine in rat. On a more general extent, this work demonstrated that the selectivity of the fragmentation pathway (MS³) can be used as alternative approach to significantly improve detection capability in complex situation (e.g., small molecules in complex matrices) rather than increasing time for sample preparation and chromatographic separation.     

GC/MS and LC/MS Phytochemical Analysis of Vigna unguiculata L. Walp Pod

Vigna unguiculata (L. Walp) or Cowpea pod methanolic extracts phytochemical analysis, total phenolic content (TPC), and secondary metabolite profiling were determined using gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) analysis. GC/MS analysis revealed twenty compounds in the extract, while LC/MS analysis identified twenty-four compounds. GC/MS chromatogram analysis suggested the presence of opioid α-N-Normethadol a major constituent found in methanolic extract and fatty acid esters carotenoid is found second major constituent. LC/MS chromatogram and the mass spectral analysis demonstrated the presence of flavonoids, carotenoids, and alkaloids as major phytochemicals. We investigated the antibacterial, anti-fungal, and anti-oxidant activity of pod methanolic extract. The extract was found equally effective against E. coli, S. pyogenes, and P. aeruginosa with MIC 100 μg/mL similar to the standard Ampicillin (MIC 100 μg/mL). C. albicans were found to be most susceptible to Vign unguiculata pods methanolic extract with a MIC of 250 μg/mL. The pod extract showed significant DPPH scavenging activity (IC₅₀=78.38±0.15) which suggests its antioxidant potential.     

MS/MS profiling of taxoids from the needles of Taxus wallichiana

Ammonium cationisation has been used for taxoid profiling of partially purified methanolic extracts of needles of Taxus wallichiana growing in different regions of the Himalayas (Kashmir, Himachal Pradesh, UP Hills, Darjeeling, Sikkim and Arunachal Pradesh) by electrospray ionisation tandem mass spectrometry (MS/MS). The MS/MS spectra of the [M + NH4]+ or [M + H]+ ions gave structurally diagnostic fragment ions which revealed information about the taxane skeleton as well as the number and nature of the substituents. The rearranged 11(15-->1)-abeo-taxanes showed a characteristic elimination of the hydroxyisopropyl group with an acetoxy/benzoyloxy group from C-9. The identification of the taxoids was achieved by comparison of the MS/MS spectra with those of authentic taxoids or was based on biogenetic grounds. The results were corroborated by liquid chromatography-MS analysis. Out of the 50 taxoids identified, 21 belonged to the rearranged class. The presence of paclitaxel in the samples from four regions was confirmed: the study also revealed the occurrence of several basic taxoids in these samples. MS/MS profiling by electrospray ionisation was shown to be a fast and reliable technique for the analysis of taxoid samples.     

The presence of 12β-deoxydecarbamoylsaxitoxin in the Japanese toxic dinoflagellate Alexandrium determined by simultaneous analysis for paralytic shellfish toxins using HILIC-LC–MS/MS

A differential screening study using high-resolution (HR)-hydrophilic interaction chromatography (HILIC)-electrospray ionization (ESI)–quadrupole time-of-flight mass spectrometry (Q-TOF MS) was conducted to identify saxitoxin (STX) analogues in the marine dinoflagellate toxic sub-clone Alexandrium tamarense Axat-2 and the non-toxic sub-clone UAT-014-009 derived from the same Japanese isolate. One unknown compound was identified only in the toxic sub-clone and was found to have the molecular formula C₉H₁₆N₆O₂. This structure differed from that of decarbamoyl STX (dcSTX; C₉H₁₆N₆O₃) by the loss of a single oxygen. A 12-deoxy-dcSTX standard (a mixture of 12α- and β-deoxy-dcSTX) was chemically prepared from dcSTX by reduction with sodium borohydride. The unknown compound in the toxic strain of A. tamarense was identified as 12β-deoxy-dcSTX by comparison of its HR-HILIC-LC–MS retention time and HR–MS/MS spectrum with those of the chemically prepared standard, and the identification was confirmed by high-sensitivity HPLC analysis with post-column fluorescent derivatization. Moreover, two Japanese isolates of A. catenella showing toxin profiles different from that of A. tamarense were also found to contain 12β-deoxy-dcSTX. Previously, 12β-deoxy-dcSTX was isolated from the freshwater cyanobacterium Lyngbya wollei, which produces a unique set of STX analogues. This study is the first evidence of the presence of 12β-deoxy-dcSTX in marine dinoflagellates.     

Traceability of Amanita fuliginea poisoning using DNA barcoding and UPLC-MS/MS

Amanita fuliginea is a lethal poisonous mushroom found in Japan and southern China. The primary toxins are α-amanitin (α-AMA) and β-amanitin (β-AMA). There is a lack of systematic and comprehensive investigations on the traceability of A. fuliginea poisoning due to technological limitations. This study aimed to examine whether A. fuliginea poisoning incidents could be traced using DNA barcoding and ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS). We collected A. fuliginea specimens and prepared cooked and cooked plus simulated gastric fluid (SGF)-treated samples. We then performed DNA barcoding of internal transcribed spacer regions for species identification and UPLC-MS/MS for toxin level determination. Our results indicate that under the experimental conditions used herein, DNA barcoding can be used for molecular identification of mushroom samples that are cooked and/or cooked plus SGF-treated for less than 30 min; UPLC-MS/MS can be used for toxin analysis of cooked and SGF-treated (0–1440 min) samples. This is the first time that DNA barcoding and UPLC-MS/MS have been combined for studying the toxicological traceability of A. fuliginea using simulated gastric contents or vomit in northern China. Our data provide support for the treatment of clinical mushroom poisoning cases.     

Bioanalytical method for the simultaneous determination of d- and l-serine in human plasma by LC/MS/MS

d-Serine is an endogenous modulator of N-methyl-d-aspartate (NMDA) receptors. Plasma concentrations of d-serine and the ratio of d-serine to total serine may be used as clinically-translatable biomarkers in NMDA receptor-related disease. We developed a highly sensitive and specific method using high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the d- and l-isomers of serine in human plasma. Since d- and l-serine are endogenous components, phosphate buffered saline was used as the surrogate matrix. d- and l-serine in human plasma and PBS were treated by cationic exchange solid phase extraction. d-Serine (m/z 106.1 > 60.0), l-serine (m/z 106.1 > 60.1) and dl-serine-d₃ (m/z 109.1 > 63.0) were detected using a multiple reaction monitoring. The enantiomer separation of d- and l-serine was successfully achieved without any derivatization step using tandemly-arranged and ice-cold CROWNPAK CR-I(+) columns with an isocratic mobile phase comprised of 0.3% trifluoroacetic acid in 10% acetonitrile. The standard curves were linear throughout the calibration range with 0.01–10 μg/mL (d-serine) and 0.1–100 μg/mL (l-serine), respectively. Intra-day and inter-day precision and accuracy of the quality control samples were within relative standard deviations of less than 15%. The endogenous concentrations of d- and l-serine in human plasma were 0.124–0.199 and 7.97–13.1 μg/mL, respectively.     

pepgrep: A Tool for Peptide MS/MS Pattern Matching

Typically, detection of protein sequences in collision-induced dissociation (CID) tandem MS (MS2) dataset is performed by mapping identified peptide ions back to protein sequence by using the protein database search (PDS) engine. Finding a particular peptide sequence of interest in CID MS2 records very often requires manual evaluation of the spectrum, regardless of whether the peptide-associated MS2 scan is identified by PDS algorithm or not. We have developed a compact cross-platform database-free command-line utility, pepgrep, which helps to find an MS2 fingerprint for a selected peptide sequence by pattern-matching of modelled MS2 data using Peptide-to-MS2 scoring algorithm. pepgrep can incorporate dozens of mass offsets corresponding to a variety of post-translational modifications (PTMs) into the algorithm. Decoy peptide sequences are used with the tested peptide sequence to reduce false-positive results. The engine is capable of screening an MS2 data file at a high rate when using a cluster computing environment. The matched MS2 spectrum can be displayed by using built-in graphical application programming interface (API) or optionally recorded to file. Using this algorithm, we were able to find extra peptide sequences in studied CID spectra that were missed by PDS identification. Also we found pepgrep especially useful for examining a CID of small fractions of peptides resulting from, for example, affinity purification techniques. The peptide sequences in such samples are less likely to be positively identified by using routine protein-centric algorithm implemented in PDS. The software is freely available at http://bsproteomics.essex.ac.uk:8080/data/download/pepgrep-1.4.tgz.     

Integration of LC/MS,NMR and Molecular Docking for Profiling of Bioactive Diterpenes from Euphorbia mauritanica L. with in Vitro Anti-SARS-CoV-2 Activity

In spite of tremendous efforts exerted in the management of COVID-19, the absence of specific treatments and the prevalence of delayed and long-term complications termed post-COVID syndrome still urged all concerned researchers to develop a potent inhibitor of SARS-Cov-2. The hydromethanolic extracts of different parts of E. mauritanica were in vitro screened for anti-SARS-Cov-2 activity. Then, using an integrated strategy of LC/MS/MS, molecular networking and NMR, the chemical profile of the active extract was determined. To determine the optimum target for these compounds, docking experiments of the active extract's identified compounds were conducted at several viral targets. The leaves extract showed the best inhibitory effect with IC₅₀ 8.231±0.04 μg/ml. The jatrophane diterpenes were provisionally annotated as the primary metabolites of the bioactive leaves extract based on multiplex of LC/MS/MS, molecular network, and NMR. In silico studies revealed the potentiality of the compounds in the most active extract to 3CLpro, where compound 20 showed the best binding affinity. Further attention should be paid to the isolation of various jatrophane diterpenes from Euphorbia and evaluating their effects on SARS-Cov-2 and its molecular targets.     

Accelerating high quality bioanalytical LC/MS/MS assays using fused-core columns

High quality, ultra-fast bioanalytical LC/MS/MS methods were developed using short columns packed with fused-core particles and high (1.0–3.0 mL/min) flow rates. For more than two years, at flow rates up to 3.0 mL/min, using 0.33 min non-ballistic gradients, these methods were shown to provide comparable or better performance than slower assays for accuracy, precision, sensitivity, specificity, and ruggedness, and met all criteria required by the bioanalytical regulatory guidance.     

LC-ESI/MS determination of xanthone and secoiridoid glycosides from in vitro regenerated and in vivo Swertia chirayita

A rapid analytical method has been developed to determine xanthone and secoiridoid glycoside in in vitro and in vivo Swertia chirayita extracts. Ultra performance liquid chromatography–electrospray ionization mass spectrometry (LC-ESI/MS) was applied and validated for the analysis of xanthone and secoiridoid glycoside a potential active component isolated from methanolic extracts of in vitro and in vivo Swertia chirayita plantlets. Chromatographic separation was achieved on a RP-C18 column using gradient elution. Mangiferin (Xanthone), Amarogentin and Swertiamarin (Secoiridoid glycosides) were identified in both the extracts. In the LC/ESI-MS spectra, major [M + H] ⁺ and [M + Na] ⁺ ions were observed in positive ion mode and provided molecular mass information. An ultra-performance liquid-chromatography in combination with electrospray ionization tandem mass spectrometry involving metal cationisation was successfully utilized for the rapid identification of xanthone and secoiridoid glycosides. This method is suitable for the routine analysis, as well as for the separation and identification of known and novel secoiridoid glycoside and xanthone.