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Mastitis, a prevalent and economically important disease in the dairy industry, poses substantial challenges to dairy cow health, milk quality, and farm profitability worldwide. Mastitis is predominantly caused by bacterial infections. The objective of this study was to estimate the sensitivity (Se) and specificity (Sp) of bacterial culture and the VetMAX™ MastiType Multi Kit PCR in identified clinical mastitis pathogens. A total of 396 quarter-level milk samples were collected from 396 cows with clinical mastitis on 29 farms in the subtropical dairy region of Australia between March and December 2021. These samples were cultured and tested by PCR, and analysed using Bayesian latent class analysis under the assumption of one population two tests and also of three populations two tests, by dividing the population into subpopulations based on regions. Informative priors used in the analysis were calculated from published evidence. Models were compared using the Deviance Information Criterion (DIC). Sensitivity analysis was performed to evaluate the impact of changes in priors. The most common isolates cultured and detected by PCR were Streptococcus uberis (17.4 % and 27.3 %, respectively) and Escherichia coli (12.6 % and 25.0 %, respectively). Under the assumption of one population two tests , the Se of PCR (at cycle threshold (Ct) ≤ 37) was higher than that of bacterial culture for both pathogens: for E. coli , the Se was 50.2 % (95 % posterior probability interval (PPI): 37.4; 74.1) for bacterial culture, and 93.7 % (95 % PPI: 85.5; 98.4) for PCR. For S. uberis , the Se was 50.4 % (95 % PPI: 40.9; 61.3) for bacterial culture, and 81.5 % (73.0; 88.9) for PCR. Conversely, the Sp of bacterial culture was higher than that of PCR for both pathogens: for E. coli , the Sp was 99.2 % (97.8; 100) for bacterial culture, and 95.1 % (87.8; 99.4) for PCR. For S. uberis , the Sp was 99.2 % (95 % PPI: 97.6; 100) for bacterial culture, and 96.7 % (95 % PPI: 92.1; 99.2) for PCR. Bayesian latent class analysis with three populations two tests was only performed for S. uberis. For E. coli, this could not be performed because there were no PCR-positive results in one subpopulation. Under the assumption of three populations two tests , for S. uberis, the Se was 49.6 % (40.6; 59.4) for bacterial culture, and 81.1 % (72.6; 88.6) for PCR; and the Sp for bacterial culture was 99.1 % (97.7; 100), and for PCR was 96.9 % (93.0; 99.3). The DIC for the one population two tests model was lower than the DIC for the three populations two tests model. The sensitivity analysis for the one population two tests model demonstrated that a 10 % reduction in priors led to substantial changes in Se of both bacterial culture and PCR tests for E. coli and S. uberis , with overlap percentages ranging from 80.6 % to 92.2 %. In contrast, the Sp of bacterial culture and PCR tests remained relatively stable despite changes in priors, except for the Sp of PCR test for E. coli. In summary, the VetMAX™ MastiType Multi Kit demonstrated higher Se compared to bacterial culture, suggesting its potential as a routine test for identifying mastitis pathogens in milk samples from cows with clinical mastitis. While the bacterial culture method offered higher Sp in pathogen detection; results obtained following bacterial culture and subsequent susceptibility testing remain valuable, particularly in guiding antimicrobial treatment for mastitis. [ABSTRACT FROM AUTHOR]

The manufacturing of indigo naturalis requires prolonged leaf soaking and lime stirring; the resulting indigo purity is less than 3.00% and the yield of indigo (measured in stems and leaves weight) is less than 0.50%, making it unsuitable for use in industrial procedures like printing and dyeing. An enzymatic method of creating indigo without the requirement for lime was investigated in order to generate high purity indigo. Single factor tests were performed to optimize the enzymatic preparation conditions. The findings showed that 60 °C, pH 5.5, 200 mL of leaves extract containing 0.45 mg/mL indican, and a 4:1 ratio of the acidic cellulose (activity: 9000 U/mL, liquid) to indican were the ideal parameters for enzymatic preparation. The yield of indigo was 40.32%, and the contents of indigo and indirubin were 37.37% and 2.30%, respectively. MALDI-TOF-MS in positive ion mode and UPLC-Q-TOF-MS in both positive and negative ion modes were used to analyze indigo extracts from Baphicacanthus cusia (Nees) Bremek by enzymatic preparation. It has been discovered that 13 alkaloids, 5 organic acids, 3 terpenoids, 3 steroids, 2 flavones, and 7 other compounds are present in indigo extracts. The presence of the indigo, indirubin, isorhamnetin, tryptanthrin, indigodole B, and indigodole C determined by UPLC-Q-TOF-MS was verified by MALDI-TOF-MS analysis. The enzymatic preparation of indigo extracts kept the same chemical makeup as conventional indigo naturalis. Thermal analysis and SEM morphology were used to confirm that there was no lime in the indigo extract. During the enzymatic process, Baphicacanthus cusia (Nees) Bremek was employed more effectively, increasing the yield and purity of indigo. • An enzymatic technique without the need for lime was investigated in order to produce high purity indigo. • The enzymatic preparation of indigo extracts kept the same chemical makeup as conventional indigo naturalis. • The indigo extract obtained through enzymatic production was a loose, inhomogeneous powder that contained 37.37% indigo. [ABSTRACT FROM AUTHOR]

In eukaryotic organisms, cell-signalling completely relies on Post Translational Modifications (PTMs) that can function as regulatory switches. Phosphorylation is a fundamental and frequently occurring PTM in almost all eukaryotes. Herein, we have studied the importance of protein phosphorylation using classical proteomic techniques in C. elegans upon bacterial infection. The differentially regulated proteins during bacterial infection were excised from SDS-PAGE (One-Dimensional) gel (TiO 2 column elutes) and subjected to MALDI-TOF-MS which ended up in identifying 220 proteins kinetically. KEGG pathway analysis of those proteins suggested that MAPK pathway was part of the innate immunity. Thus, we have characterized p38-MAPK (one of the crucial downstream MAPKs) using immunoblotting, subcellular fractionation, coimmunoprecipitation, LC-MS/MS, bioinformatics studies and qPCR. Meanwhile, KU25 strain (pmk-1 mutant) exhibited an earlier mortality during infection suggesting the crucial role of p38-MAPK during host-pathogen interaction. Interestingly, Reactome pathway analysis of p38 interactors (CoIP coupled to LC-MS/MS) revealed the involvement of various proteolytic pathway players (ubiquitination, SUMOylation and Neddylation) during bacterial infection. Further, the regulation of SUMOylation and Neddylation was identified and validated using immunoblotting and qPCR analyses, respectively. Concisely, our study indicated that bacterial infection triggers the MAPK cascade to elicit innate immunity which in turn recruits proteolytic pathways to counteract the invading pathogen. [Display omitted] • MAPK pathway is required to combat the bacterial exposure • p38 localizes into nucleus during bacterial exposure • LC-MS/MS analysis reveals the interacting partners of p38 during bacterial exposure • C. elegans innate immunity requires proteolysis pathway(s) during bacterial invasion • This study discloses the role of proteolysis pathway along with p38-MAPK during bacterial exposure [ABSTRACT FROM AUTHOR]

Honey is the most famous natural sweet substance produced by honeybees (Apis mellifera). It contains numerous functional compounds, from which health benefits are obtained. In Chile, the production of honey is associated with its unique biodiversity, but it is exported mostly, as bulk honey. This work aimed to characterize the chemical and biological properties of Chilean honey on a large scale. The use of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) technique, combined with multivariate statistical analysis was introduced to study the chemical profiles of polyfloral honey. The use of a complementary mass spectrometry method allowed the identification of 25 different constituents in the studied honey, including hydrocarbons, acids, esters, glycoside isoprenoids, ketones, and a dihydroxyflavanone. The evaluation of biological properties in Chilean honey was measured in a representative number of polyfloral samples. For this purpose, the total phenolics and flavonoid content were measured in the selected samples. The antioxidant activity of the honey was evaluated through the ferric reducing antioxidant power assay (FRAP) and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method. On the other hand, the antibacterial activity was assessed by the dilution technique to determine the minimum inhibitory concentration (MIC) of the honey against seven bacterial strains, including Gram-positive and Gram-negative bacteria. The results demonstrated that the antioxidant and antibacterial activities of Chilean honey are variable and they could have similar properties in comparison with other well-recognized bioactive honey. [ABSTRACT FROM AUTHOR]

• Endophytic microorganisms have been considered a valuable biotechnological tool. • Endophytic from cacti are acknowledged to be a source of bioactive compounds. • Endophytic bacteria from Cereus peruvianus plants were retrieved in the current study. • Endophytic bacteria were isolated from in vivo and in vitro cultivated plants. Somaclones were indicated as the most promising source of endophytic bacteria. Bioprospecting of endophytic microorganisms has been considered a promising resource for obtaining new biomolecules with biotechnological applications. Since few studies are available on the diversity of endophytic bacteria associated with species of the Cactaceae family, current analysis isolates and characterizes the communities of endophytic bacteria associated with the species of C. peruvianus grown in vivo obtained from seed and with somaclone of the same species regenerated in vitro to prospect for their plant-growth promoting activity. Isolation of endophytic bacteria from plants obtained from seeds and from regenerated somaclone in vitro identified nine species of endophytic bacteria (Moraxela osloensis, Corynebacterium mycetoides, Micrococcus luteus, Kocuria palustris, Staphylococcus epidermidis, Staphylococcus cohnii, Pseudomonas stuzeri, Bacillus licheniformis and Microbacterium sp.) not previously described in cacti, and signalized the somaclones as the most promising source of endophytic bacteria for obtaining new biomolecules with biotechnological applications. Somaclones are indicated as the most promising source of endophytic bacteria due to their high concentration of endophytes per gram identified in fresh somaclone tissue, the identification of the highest proportion endophytic bacteria, a source of IAA, and with the highest capacity of biological nitrogen fixation, and EPS production exclusively by the endophytic bacteria isolated from the in vitro regenerated somaclone. [ABSTRACT FROM AUTHOR]

By employing the mixed matrix, we established a new MALDI-TOF MS platform for rapid and accurate identification of clinical microorganisms. [Display omitted] • A cooperative matrix was demonstrated more peaks in the analysis of bacteria. • Much more components presented in the bacterial colony can be easily identified. • The characteristic peaks identified can directly distinguish similar bacteria. • The present method is simple, rapid, reproducible and accurate. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is emerging as the most powerful tool for the identification of bacteria. Herein, we demonstrated that the capability of MALDI-TOF-MS towards the discrimination of clinical bacteria can be further dramatically improved. A cooperative matrix consisting of (E)-propyl α-cyano-4-hydroxylcinnamylate (CHCA-C3) and α-cyano-4-hydroxycinnamic acid (CHCA) was used to replace the commonly used matrix CHCA. As a result, 1.4–3.8 times more fingerprint peaks have been identified by the cooperative matrix in comparison with CHCA when six different bacteria were studied. The peaks distribute in the wider m / z range, and the spectral quality has also been significantly improved. As an example of practical applications, the direct and more reliable discrimination between Escherichia coli (E. coli) and Shigella sonnei (S. sonnei) was achieved based on their protein fingerprints, which was previously claimed to be impossible by CHCA. Thus, a promoted MALDI-TOF-MS platform towards bacterial identification has been established by utilizing the new cooperative matrix, which will exhibit great potential for many applications. [ABSTRACT FROM AUTHOR]

The formaldehyde-based adhesive is commonly used in wood industry; however, the emission of formaldehyde is a fatal defect, which endangers people's health as well as pollutes the environment. Herein, the non-toxic and low-volatility glyoxal (G) is chosen to react with dimethylolurea (DMU) to prepare a novel and excellent water-resistant wood adhesive of glyoxal-dimethylolurea (G-DMU) resin. Quantum chemical calculations, structure characterizations, and performance measurements are carried out to study the reaction mechanism, and structure-performance relations of G-DMU resin with different DMU/G molar ratios. The results show that during the synthesis of G-DMU resin, the hydroxyl compounds N-p-G-DMU and N-p-G1-DMU, as well as the carbocation intermediates C-p-G-DMU and C-p-G1-DMU are formed by the addition reactions between DMU and protonated glyoxal (p-G and p-G1). The plywood bonded with G-DMU resin with the optimum DMU/G molar ratio of 1.2:1.0 has the best water resistance. Its wet shear strength is increased by 145% (cold water) and 193% (63 °C) over the DMU/G molar ratio of 0.8: 1.0. The analyses of structure characterizations reveal that the generation of C=N, C–O–C, N–C–N, and heterocyclic rings, simultaneously, the main cross-linked components concentratedly are characterized in 400–500 Da, 500–550 Da, 698 Da, and 712 Da regions. In addition, the high molecular weight oligomers at 658–712 Da, 874 Da, 1037 Da and 1213 Da promote the formation of a highly cross-linked network during the curing process. Meanwhile, these results are in good agreement with the results of quantum chemical calculations. Thus, this work establishes the theoretical foundation for the novel glyoxal-based resin of G-DMU resin. [Display omitted] • A novel eco-friendly glyoxal-dimethylolurea (G-DMU) resin has been successfully prepared. • The molar ratio DMU/G is a key parameter to influence the properties of G-DMU resin. • N-p-G-DMU, N-p-G1-DMU, C-p-G-DMU and C-p-G1-DMU are key intermediates. • The addition reactions of G or G1 with DMU are thermodynamically unfavorable. • The high molecular weight oligomers promotes a highly cross-linked network. [ABSTRACT FROM AUTHOR]

Toxic metal contamination in soils due industrialization is nowadays a concern to the scientists worldwide. The current study deals with the evaluation of response and tolerance by isolated metallophilic bacteria in different lead concentrations (100 ppm to 1000 ppm). By taking optical densities of the isolates, the minimum inhibitory concentration (MIC) of Pb2+ were determined.16S rRNA and MALDI-TOF MS were used for the identification of the bacteria. Total of 37 isolates were observed, among them 04 (Staphylococcus equorum, Staphylococcus warneri , Bacillus safensis and Bacillus thuringiensis), isolated were detected having efficacy of Pb2+tolerance and sequestration at varying MIC. Furthermore, B. thuringiensis was observed to have highest (900 ppm) tolerance for lead and lowest (500 ppm) for Staphylococcus warneri. Moreover, the highest (65.3%) sequestration potential has been observed for B. thuringiensis and least (52.8%) for S. warneri. The tolerance and sequestration potential properties of these isolated species can be utilised to exterminate heavy metals and reduce their toxicity from the contaminated environment. [ABSTRACT FROM AUTHOR]

Involvement of several candidate immune regulatory players at transcriptomic levels during microbial interactions were reported by involving C. elegans as a model system for the past few years. In the present study, we have identified a wide range of phenotypical, physiological and biochemical alterations in C. elegans triggered due to S. Typhi infection using standard approaches. We performed several behavioural studies and molecular studies such as liquid-phase IEF, MALDI-MS and bioinformatics analyses. S. Typhi exerted a slow killing against C. elegans and prompted several phenotypical changes such as egg laying defects, pharyngeal arresting, and triggered functional group variations which were disclosed using FT-IR. Proteome analysis using liquid phase IEF and MALDI-ToF-Mass Spectrometry ended up with the identification of 123 proteins which contains human orthologs. Bioinformatics analysis of the MS identified proteins revealed the involvement of ubiquitination pathway which was then validated using immunoblotting. Extensive studies similar to our study with the utility of high-throughput OMICS technologies during host pathogen interactions may pave a way for the identification of biomarkers against bacterial diseases. Unlabelled Image • Comprehensive analysis of bacterial exposure in C. elegans reveals the multifaceted physiological and biochemical abnormalities. • This is one of the few studies to utilize MicroRotofor™ coupled with MALDI-MS for understanding host pathogen interactions. • The orthologous genes of MS identified C. elegans proteins are conserved in H. sapiens , D. melanogaster , M. musculus, etc. • Immune regulatory proteins identified in the study have many reported glycosylated PTM sites. [ABSTRACT FROM AUTHOR]

Mycolicibacter kumamotonensis (M. kumamotonensis), formerly Mycobacterium kumamotonense , is a nontuberculous mycobacteria species, which was first separated from Mycobacterium terrae complex in 2006. Reports about infections caused by M. kumamotonensis are extremely rare, with most of them being lung infection. Here, we report the case of a 68-year-old man with a hobby of gardening who developed swelling in his right middle finger. He underwent surgical debridement at a previous hospital and was diagnosed with nontuberculous mycobacteria infection based on positive findings of acid-fast staining of pus obtained from the surgical specimen. He was treated with rifampicin, ethambutol, and clarithromycin, but the swelling worsened. Therefore, he was referred to our hospital for further examination and treatment. We performed a second debridement and added isoniazid to the treatment regimen, but the swelling continued to worsen. We then administered levofloxacin, but his condition did not change. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and DNA sequencing analysis confirmed M. kumamotonensis as the causative bacterium. Since the finger swelling did not improve, the patient underwent a third debridement and amikacin was added to the treatment regimen. Finally, the infection was controlled. He completed amikacin therapy and will continue treatment with the other five antibiotics for a total of 24 months. To the best of our knowledge, this is the first report of a patient with M. kumamotonensis soft tissue infection. We consider this case might provide important insights into the diagnosis and treatment of soft tissue infections caused by M. kumamotonensis. [ABSTRACT FROM AUTHOR]

Obligate anaerobes exist as resident flora in various sites in humans, but they are also emphasized as endogenous causative microorganism of infections. We performed surveillance to understand the trend of drug susceptibility in obligate anaerobic bacteria in the Kinki area of Japan. In the experiment, we used 156 obligate anaerobe isolates collected from 13 institutions that participated in the Study of Bacterial Resistance Kinki Region of Japan. MALDI Biotyper was used to identify the collected strains, and among the 156 test strains, those that could be identified with an accuracy of Score Value 2.0 or more included 6 genera, 30 species, and 144 strains (Bacteroides spp. 77 strains, Parabacteroides sp. 2 strains, Prevotella spp. 29 strains, Fusobacterium spp. 14 strains, Porphyromonas spp. 2 strains, and Clostridioides difficile 20 strains), and they were assigned as subject strains for drug susceptibility testing. The drug susceptibility test was carried out by broth microdilution method using Kyokuto Opt Panel MP ANA (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) and judged according to CLSI criteria. As a result, Bacteroides and Parabacteroides species showed good sensitivities to tazobactam-piperacillin, imipenem, metronidazole and chloramphenicol, and low sensitivities to ampicillin, cefoperazone and vancomycin. Prevotella species showed good sensitivities to sulbactam-ampicillin, tazobactam-piperacillin, cefmetazole, imipenem, doripenem and metronidazole. Susceptibility rates to other drugs were slightly different depending on the bacterial species. Both Fusobacterium spp. and Porphyromonas spp. showed high sensitivities to many drugs. C. difficile was highly sensitive to vancomycin and metronidazole, having MIC 90 s of 0.5 μg/mL and ≤2 μg/mL, respectively. [ABSTRACT FROM AUTHOR]

We report herein a rationally designed pyrene linked substrate for quantitative protease activity assay via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In this proof-of-concept study, a trypsin-specific peptide with the sequence of GGGGRG was selected to conjugate with pyrene forming a pyrene linked peptide probe, Py-GGGGRG. In the presence of trypsin, the Py-GGGGRG probe can be specifically hydrolyzed into Py-GGGGR. The introduction of pyrene greatly increased ionization efficiency of Py-peptides, and Py-peptides could be selectively captured from complex mixtures by a facially fabricated polystyrene coated MALDI plate through hydrophobic and π-π stacking interactions. As a result, trypsin activity can be directly quantified by relative intensity ratio of product and substrate via MALDI-TOF-MS without the use of external internal standard. A linear range of 0.1–10 μg/mL and a relatively low detection limit of 29 ng/mL were obtained. This method has also been successfully used for quantification of trypsin activity in urine and screening the inhibitors of trypsin. Besides, the proposed strategy was also validated for another protease, chymotrypsin, by using the probe Py-GGGGGGYG. Therefore, owing to simplicity, high-throughput capacity and quantificational accuracy, the proposed method shows great potential for activity assay of various proteases and screening their inhibitors via application of specific peptide sequences. fx1 • A rationally designed pyrene linked peptide probe was used for quantitative protease assay by MALDI-TOF-MS. • The incorporation of pyrene greatly increased the ionization efficiency of probe and enabled the on-line purification. • This proposed method was successfully used for quantification of trypsin assay in urine and screening its inhibitors. • This proposed method was simple, high-throughput and had great quantitative accuracy. • As a proof-of-concept study, this proposed approach also can be used for other protease assay. [ABSTRACT FROM AUTHOR]

Highlights • 90% of zinc protoporphyrin IX is protein-bound at 0 h of extract-incubation. • The share of unbound zinc protoporphyrin IX rises from 10% to 50% within 72 h. • An unknown fluorescence peak was detected at a protein mass of >100 kDa. Abstract Zinc protoporphyrin IX (ZnPP) is known to accumulate in most meat products during storage. However, the pathway of its formation is not yet completely clarified. To gain more insights into the specificity of ZnPP occurrence, a SEC -HPLC-UV-fluorescence setup was established to screen the proteins in aqueous meat extracts for their ZnPP fluorescence during incubation. In accordance with previous studies it was identified by SDS-PAGE and MALDI-TOF-MS that ZnPP formation takes place in myoglobin. In this study, valuable new insights into the ZnPP forming pathway were gained, as our results indicated that a significant part of ZnPP – after being formed within the protein – is transitioned into free ZnPP during incubation. Additionally, the obtained results implied that ZnPP may also occur in proteins of higher molecular weight (>100 kDa). [ABSTRACT FROM AUTHOR]

Abstract We report a facile method for detection of metallothionein (MT), a promising clinically relevant biomarker, in spiked plasma samples. This method, for the first time, integrates molecularly imprinted polymers as purification/pretreatment step with matrix assisted laser desorption/ionization time-of-flight mass spectrometric detection and with laser ablation inductively coupled plasma mass spectrometry for analysis of MTs. The prepared MT-imprinted polydopamine layer showed high binding capacity and specific recognition properties toward the template. Optimal monomer (dopamine) concentration was found to be 16 mM of dopamine. This experimental setup allows to measure µM concentrations of MT that are present in blood as this can be used for clinical studies recognizing MT as marker of various diseases including tumour one. Presented approach not only provides fast sample throughput but also avoids the limitations of methods based on use of antibodies (e.g. high price, cross-reactivity, limited availability in some cases, etc.). Graphical abstract fx1 Highlights • Molecularly imprinted polymers as purification method for MT-1G was firstly used. • The conditions for the polymers preparation were optimized. • The purification step was then followed by MALDI-TOF mass spectrometry. • The concept was tested on the spiked blood serum. [ABSTRACT FROM AUTHOR]

Abstract Seawater reverse osmosis (SWRO) is a commonly used desalination technique owing to its lesser environmental and economic impacts as compared to thermal desalination techniques. Antiscalants are used in SWRO to reduce membrane scaling caused by the supersaturation of salts present in feed water. However, to remain effective in reducing membrane scaling, antiscalants should be highly stable and resistant to biological degradation by seawater microorganisms. In this research, several bacteria from Qatar's seawater were isolated and screened for their ability to use antiscalants as a carbon and energy source. The biodiversity of antiscalant degrading seawater bacteria was demonstrated through combining the techniques of MALDI-TOF MS and principle component analysis. It was found that the bacteria isolated from Qatar's seawater such as H. aquamarina, H. elongata, P. fragi, P. stutzeri and others can degrade antiscalants and use them as a carbon and energy source. It was observed that the growth rates varied based on the type of antiscalant and the bacteria used. Among the tested strains, H. aquamarina, which is also known for its potential to cause biofouling, demonstrated the highest growth rates in antiscalants media. Thus, it was concluded that there is wide variety of bacteria in Qatar's seawater that can biodegrade the antiscalants; reducing their efficiency to combat membrane scaling. Since, these antiscalants will be used as a source of carbon and energy, microbial growth will increase resulting in enhanced membrane biofouling in SWRO. Graphical abstract Unlabelled Image Highlights • Antiscalants are commonly used to reduce scaling in seawater reverse osmosis. • The antiscalants are prone to enhance membrane biofouling. • There is a biodiversity of seawater bacteria that can biodegrade antiscalants. • Bacterial biodiversity can be explored through combining MALDI-TOF MS with PCA. • The specific growth rates vary with the type of bacteria and antiscalants. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Optimization of culture condition of tannase from Enterobacter cloacae. • Tannase from E. cloacae was purified and characterized. • The molecular weight of purified tannase was ∼45 kDa and its activity was analyzed by zymogram. • The purified tannase was analyzed by MALDI-TOF-MS, FT-IR and circular dichroism (CD) and the enzymatic reaction product was analyzed by HPLC. • The enzyme is stable over a good range of temperature and pH. Abstract Tannase production by Enterobacter cloacae strain 41 was investigated under submerged fermentation which was optimized at various circumstances such as pH, temperature, substrate, and agitation, carbon, and nitrogen sources. Tannase was purified by a two-step approach comprising of ion exchange and size exclusion chromatography, respectively. The maximum tannase production was achieved at 1.0% tannic acid concentration, incubation temperature of 50 °C, and initial pH 6.0. The molecular weight of purified tannase was 45 kDa on 10% SDS-PAGE, and it was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). The enzymatic products of purified tannase were characterized by HPLC, TLC and FT-IR spectroscopy which showed the functional groups such as OH, CO, and CC. The purified tannase retained the activity up to 90% under the condition at 50 °C and pH 6.0 after 1 h incubation. Enzyme kinetics and inhibition studies were also investigated. Cytotoxicity studies showed that the tannase has no cytotoxic effects on Vero cell line. The results indicated the E. cloacae strain 41 would give a potential source for the efficient production of tannase and can be used in tannery effluent degradation, food, and pharmaceutical industrial applications. [ABSTRACT FROM AUTHOR]

Abstract Background Little is known about enzymatic N- glycosylation in type 2 diabetes, a common posttranslational modification of proteins influencing their function and integrating genetic and environmental influences. We sought to gain insights into N -glycosylation to uncover yet unexplored pathophysiological mechanisms in type 2 diabetes. Methods Using a high-throughput MALDI-TOF mass spectrometry method, we measured N- glycans in plasma samples of the DiaGene case-control study (1583 cases and 728 controls). Associations were investigated with logistic regression and adjusted for age, sex, body mass index, high-density lipoprotein-cholesterol, non-high-density lipoprotein-cholesterol, and smoking. Findings were replicated in a nested replication cohort of 232 cases and 108 controls. Results Eighteen glycosylation features were significantly associated with type 2 diabetes. Fucosylation and bisection of diantennary glycans were decreased in diabetes (odds ratio (OR) = 0.81, p = 1.26E-03, and OR = 0.87, p = 2.84E-02, respectively), whereas total and, specifically, alpha2,6-linked sialylation were increased (OR = 1.38, p = 9.92E-07, and OR = 1.40, p = 5.48E-07). Alpha2,3-linked sialylation of triantennary glycans was decreased (OR = 0.60, p = 6.38E-11). Conclusions While some glycosylation changes were reflective of inflammation, such as increased alpha2,6-linked sialylation, our finding of decreased alpha2,3-linked sialylation in type 2 diabetes patients is contradictory to reports on acute and chronic inflammation. Thus, it might have previously unreported immunological implications in type 2 diabetes. General significance This study provides new insights into N -glycosylation patterns in type 2 diabetes, which can fuel studies on causal mechanisms and consequences of this complex disease. Highlights • Plasma protein N -glycans associate with type 2 diabetes and its risk factors • Diabetes associates with higher alpha2,6-linked and lower alpha2,3-sialylation • These sialylation changes indicate changes in pro- and anti-inflammatory processes. • We identified potential glycoprotein targets for future mechanistic studies [ABSTRACT FROM AUTHOR]

Capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) has long been proven to have excellent performance in the analysis and characterization of therapeutic proteins. However, it is rarely used for the detection of low-molecular-weight proteins or peptides. Our research has proved the ability of CE-SDS to characterize the purity of low-molecular-weight proteins (i.e., <10 kDa) and even polypeptides. In this article, insulin glargine was used as a model protein, and CE-SDS was used to analyze the samples damaged by heating and light exposure. The monomers, dimers, and trimers of insulin glargine were effectively separated, and the results of the mass spectrometry also confirmed the existence of two kinds of insulin aggregates. For comparison, the size-exclusion high-performance liquid chromatography (SE-HPLC) only showed a single aggregate peak. In addition, the denaturation conditions caused only the covalent aggregates to appear in the CE-SDS analysis. These advantages also make CE-SDS an excellent supplementary technology to the traditional SE-HPLC, providing biopharmaceutical analysts with more information. • Low-molecular-weight protein aggregation was first characterized by CE-SDS. • Insulin glargine aggregation was compared between CE-SDS and SE-HPLC. • CE-SDS achieved higher resolution and distinguished covalent from noncovalent aggregates. [ABSTRACT FROM AUTHOR]

Abstract Fresh meat harbors autochthonous microbiota with unknown risk potential, which is introduced in raw fermented sausages. Their growth can be limited by the use of safe, competitive starter strains. In the lack of time and cost-effective methods to track those starters at strain level, their assertiveness upon meat fermentation is widely unknown. Lactobacillus (L.) sakei and L. curvatus, which can be isolated from a variety of habitats, are frequently used as starter cultures. We monitored the assertiveness of 9 L. sakei and 9 L. curvatus strains in a model fermentation using MALDI-TOF-MS. An "in-house" MALDI-TOF-MS database with sub-proteome spectra of L. sakei and L. curvatus strains, as well as members of the autochthonous, spontaneously growing meat microbiota was established, validated and recognition rates were determined for each L. curvatus and L. sakei strain used. Competition studies were performed with standardized sausage batter, which was inoculated with a total of 106 cells of sets of 4–5 strains each of L. sakei and L. curvatus and 106 Staphylococcus carnosus ssp. carnosus cells. The pH and redox potential were monitored continuously. On days 0, 2 and 5 samples were taken to determine the CfU/g and a total of 96 isolates per sample were identified via MALDI-TOF-MS. MALDI-TOF-MS generally proved suitable for identification of isolates on strain level within the starter sets employed, but the recognition rate varied depending on the strain. Competition studies revealed dominance or co-dominance of strains within each set. However, their assertiveness significantly depended on the composition of the strain sets. Still, co-dominance or cooperation appeared effective to outgrow other members of the autochthonous meat microbiota, rather than dominance of single strains. For the latter, the ability to produce bacteriocins suggested itself for a crucial role in the assertiveness of starter strains. [ABSTRACT FROM AUTHOR]

Coronatine (COR) is a phytotoxin produced by Pseudomonas syringae . Its structure is similar to those of jasmonates (JAs), which play diverse roles in multiple plant biotic and abiotic defenses. However, the biological activity of COR is 1000 times greater than the activity of JA. In addition to being involved in the JA pathway, COR affects plant photosynthetic efficiency. In this study, we examined wheat blade pretreatment with COR. Blades treated with COR remained green longer than those of control plants under drought stress conditions, resulting in less yield loss with COR treatment. To investigate the mechanism of COR in drought resistance further, we employed two-dimensional gel electrophoresis technology and matrix-assisted laser desorption/ionization mass spectrometry to sequester and identify key proteins. Six COR-inducible proteins that are located in the chloroplast and involved directly in photosynthesis were found. The wheat homologue of protein gi|326509937 is degradation of periplasmic proteins 1 (DEGP1) in Arabidopsis , which is a response to photosystem II reparation, and was maintained at a low level with COR treatment. Finally, we measured levels of chlorophyll and photosynthetic performance to reveal the phenotypic effect of COR. Taken together, the results demonstrate that COR enhances drought tolerance by maintaining high photosynthetic performance. [ABSTRACT FROM AUTHOR]

Background & Aims Biomarkers are needed for early detection of Crohn’s disease (CD) and ulcerative colitis (UC) or to predict patient outcomes. Glycosylation is a common and complex posttranslational modification of proteins that affects their structure and activity. We compared plasma N -glycosylation profiles between patients with CD or UC and healthy individuals (controls). Methods We analyzed the total plasma N -glycomes of 2635 patients with inflammatory bowel diseases and 996 controls by mass spectrometry with a linkage-specific sialic acid derivatization technique. Plasma samples were acquired from 2 hospitals in Italy (discovery cohort, 1989 patients with inflammatory bowel disease [IBD] and 570 controls) and 1 medical center in the United States (validation cohort, 646 cases of IBD and 426 controls). Sixty-three glycoforms met our criteria for relative quantification and were extracted from the raw data with the software MassyTools. Common features shared by the glycan compositions were combined in 78 derived traits, including the number of antennae of complex-type glycans and levels of fucosylation, bisection, galactosylation, and sialylation. Associations of plasma N -glycomes with age, sex, CD, UC, and IBD-related parameters such as disease location, surgery and medication, level of C-reactive protein, and sedimentation rate were tested by linear and logistic regression. Results Plasma samples from patients with IBD had a higher abundance of large-size glycans compared with controls, a decreased relative abundance of hybrid and high-mannose structures, lower fucosylation, lower galactosylation, and higher sialylation (α2,3- and α2,6-linked). We could discriminate plasma from patients with CD from that of patients with UC based on higher bisection, lower galactosylation, and higher sialylation (α2,3-linked). Glycosylation patterns were associated with disease location and progression, the need for a more potent medication, and surgery. These results were replicated in a large independent cohort. Conclusions We performed high-throughput analysis to compare total plasma N -glycomes of individuals with vs without IBD and to identify patterns associated with disease features and the need for treatment. These profiles might be used in diagnosis and for predicting patients’ responses to treatment. Graphical abstract [ABSTRACT FROM AUTHOR]

Silver nanoparticles (AgNPs) loaded ammonium exchanged mordenite (NH 4 M20) were found to be an efficient inorganic matrix (AgNPs-NH 4 M20) to detect short n-alkanes, which are hardly ionized in conventional MALDI mass spectrometry. Silver ion adduct with alkanes, including n-heptadecane (C 17 H 34 ), n-nonadecane (C 19 H 40 ), n-tricosane (C 23 H 48 ), n-tetracosane (C 24 H 50 ), n-heptacosane (C 27 H 56 ), allowing the analysis of short-chain n-alkanes by MALDI-TOF-MS. Zeolite as the supporter can enhance the peak intensity of Ag + adducted n-alkanes by utilizing laser energy efficiently. The character of zeolite in AgNPs-NH 4 M20 matrix and the way of formation of Ag + adducted n-alkanes were also discussed and studied by using the quantum chemical calculation. [ABSTRACT FROM AUTHOR]

Phosphorylated peptides are important for understanding the processes of biological regulation. However, the detection of phosphopeptides remains a challenge because of their low abundance and the suppression by non-phosphopeptides. Here, we report a strategy for the facile and rapid fabrication of TiO 2 -nanoparticle-packed microchannel-array glass microchips (TMA-microchips) for in-tube solid-phase microextraction (IT-SPME) using a plasma-assisted method for the precise alignment of the microstructure. This proposed strategy was applied to the selective enrichment of phosphopeptides from a protein digestion mixture, demonstrating the high capacity and selectivity of the SPME microchips. An important feature of this array design is that it fully exploits the advantage of nanoparticles for improving extraction capacity and simultaneously provides an effective way to reduce the pressure for driving solutions; thus, it paves the way for future methods that simultaneously take advantage of nanomaterials and microchips. [ABSTRACT FROM AUTHOR]

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A facile method for foodborne bacterial enrichment and direct identification from milk samples using Janus magnetic mesoporous silica nanoparticles (Janus M-MSNs) is reported. The fluorescent-magnetic nanorods are fabricated using the one-pot sol-gel process, followed by modification with Escherichia coli antibodies to form fluorescent Janus M-MSNs (Janus M-MSNs-Ab). The Janus M-MSNs-Ab combine the fast magnetic response capacity of the magnetic heads with the strong fluorescence and bacterial-targeting properties of the silica bodies, which results in selective capture and fluorescence identification of E. coli from milk samples containing mixed bacteria. After magnetic separation, complexes of the bacteria and nanoparticles are detected through direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which has a strong, anti-interference performance. Thus, the Janus M-MSNs-Ab synthesized in this study show great sensitivity and effectivity in foodborne bacterial capture and is a promising tool for rapid bacterial detection and investigation when combined with MALDI-TOF-MS. [ABSTRACT FROM AUTHOR]

The aim of this study was to identify specific chemical bonds and characteristic structures in melanoidins formed from d -glucose and l -alanine between 130 and 200 °C. The results might be used to control the type and amount of melanoidin produced during food processing. For this purpose, complementary techniques, such as FTIR, NMR, EPR, and MALDI-ToF, were employed. At 160 °C color, solubility and UV/Vis absorption change characteristically and consequently, structural transformations could be observed in FTIR and NMR spectra. For example, sharp signals of N-H, C-N, and C-H oscillations in the l -alanine spectrum are prone to inhomogeneous broadening in melanoidins prepared above 150 °C. These changes are caused due to formation of heterogeneous macromolecular structures and occur during condensation reactions that lead to an increasing loss of water from the melanoidins with increasing temperatures. Additionally, MALDI-ToF-MS indicates the polymerization of glyoxal/glyoxylic acid and EPR shows the formation of radical structures. [ABSTRACT FROM AUTHOR]

This study aimed to assess the benefits of dithiothreitol (DTT)-based sample treatment for protein equalization to assess potential biomarkers for bladder cancer. The proteome of plasma samples of patients with bladder carcinoma, patients with lower urinary tract symptoms (LUTS) and healthy volunteers, was equalized with dithiothreitol (DTT) and compared. The equalized proteomes were interrogated using two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry. Six proteins, namely serum albumin, gelsolin, fibrinogen gamma chain, Ig alpha-1 chain C region, Ig alpha-2 chain C region and haptoglobin, were found dysregulated in at least 70% of bladder cancer patients when compared with a pool of healthy individuals. One protein, serum albumin, was found overexpressed in 70% of the patients when the equalized proteome of the healthy pool was compared with the equalized proteome of the LUTS patients. The pathways modified by the proteins differentially expressed were analyzed using Cytoscape. The method here presented is fast, cheap, of easy application and it matches the analytical minimalism rules as outlined by Halls. Orthogonal validation was done using western-blot. Overall, DTT-based protein equalization is a promising methodology in bladder cancer research. [ABSTRACT FROM AUTHOR]

Background and objective 2D-gel electrophoresis is widely used in combination with MALDI-TOF mass spectrometry in order to analyze the proteome of biological samples. For instance, it can be used to discover proteins that are differentially expressed between two groups (e.g. two disease conditions, case vs. control, etc.) thus obtaining a set of potential biomarkers. This procedure requires a great deal of data processing in order to prepare data for analysis or to merge and integrate data from different sources. This kind of work is usually done manually (e.g. copying and pasting data into spreadsheet files), which is highly time consuming and distracts the researcher from other important, core tasks. Moreover, engaging in a repetitive process in a non-automated, handling-based manner is prone to error, thus threatening reliability and reproducibility. The objective of this paper is to present S2P, an open source software to overcome these drawbacks. Methods S2P is implemented in Java on top of the AIBench framework, and relies on well-established open source libraries to accomplish different tasks. Results S2P is an AIBench based desktop multiplatform application, specifically aimed to process 2D-gel and MALDI-mass spectrometry protein identification-based data in a computer-aided, reproducible manner. Different case studies are presented in order to show the usefulness of S2P. Conclusions S2P is open source and free to all users at http://www.sing-group.org/s2p . Through its user-friendly GUI interface, S2P dramatically reduces the time that researchers need to invest in order to prepare data for analysis. [ABSTRACT FROM AUTHOR]

Pigment-binding proteins play important roles in crustacean shell colour change. In this study, a red colour-related pigment-binding protein, designated LvPBP75, was purified from the shell of Litopenaeus vannamei . HPLC and PAGE analysis showed that LvPBP75 was a homogeneous monomer with molecular mass of 75 kDa. Peptide mass fingerprint analysis revealed that LvPBP75 belonged to hemocyanin, and the released pigment from heated LvPBP75 showed a λ max at 481 nm in acetone. The significant red-colour change temperatures were detected at 30 and 80 °C, respectively. Based on the determined amino acid fragments, a full-length cDNA of LvPBP75 was cloned and sequenced. The ORF encodes a protein of 662 amino acids having 80% identity with penaeidae hemocyanin. These results strongly suggest a novel function of hemocyanin, namely binding with pigment, and its involvement in L. vannamei shell colour change. [ABSTRACT FROM AUTHOR]

N-glycan profiling is a powerful approach for analyzing the functional relationship between N-glycosylation and cancer. Current methods rely on either serum or fresh tissue samples; however, N-glycan patterns may differ between serum and tissue, as the proteins of serum originate from a variety of tissues. Furthermore, fresh tissue samples are difficult to ship and store. Here, we used a profiling method based on formalin-fixed paraffin-embedded (FFPE) tissue sections from lung adenocarcinoma patients. We found that our method was highly reproducible. We identified 58 N-glycan compositions from lung adenocarcinoma FFPE samples, 51 of which were further used for MS n -based structure prediction. We show that high mannose type N-glycans are upregulated, while sialylated N-glycans are downregulated in our FFPE lung adenocarcinoma samples, compared to the control samples. Our receiver operating characteristic (ROC) curve analysis shows that high mannose type and sialylated N-glycans are useful discriminators to distinguish between lung adenocarcinoma and control tissue. Together, our results indicate that expression levels of specific N-glycans correlate well with lung adenocarcinoma, and strongly suggest that our FFPE-based method will be useful for N-glycan profiling of cancer tissues. Significance Glycosylation is one of the most important post-translational protein modifications, and is associated with several physiopathological processes, including carcinogenesis. In this study, we tested the feasibility of using formalin-fixed paraffin-embedded (FFPE) tissue sections to identify changes in N-glycan patterns and identified the differentially expressed N-glycans of lung adenocarcinoma. Our study shows that the FFPE-based N-glycan profiling method is useful for clinical diagnosis as well as identification of potential biomarkers, and our data expand current knowledge of differential N-glycan patterns of lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Objectives To assess the potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in rapid identification of bacteria from smear-positive cerebrospinal fluid (CSF) in a cohort of patients with meningitis. Methods Single-centre observational study, including adults and children with community-acquired or postneurosurgical bacterial meningitis. Meningitis was defined using established criteria. Samples of CSF that had a positive CSF Gram stain were directly examined by MALDI-TOF-MS. Identification was considered accurate when identical to the CSF culture or PCR results (species and genus level). Laboratory workers performing the MALDI-TOF-MS and interpreting its results were blinded to the direct smear results, except for the fact that it was positive. MALDI-TOF-MS results were not conveyed to clinicians. Results MALDI-TOF-MS was tested on 44 CSF samples; ten samples were obtained from patients with community-acquired meningitis, and 34 samples were from patients with postneurosurgical meningitis. The assay identified bacteria correctly in 17/21 of the samples with Gram-negative rods observed on the direct smear, all obtained from patients who had undergone neurosurgery, (sensitivity 81%, 95% CI 64.2%–97.7%). In the postneurosurgical group, Gram-positive cocci were identified correctly in only 1/11 (9.1%) of the samples, and Candida species were not identified in two samples. Among patients with community-acquired meningitis, the assay did not identify Streptococcus pneumoniae in eight of eight samples, Neisseria meningitidis in one sample (1/1), and Streptococcus agalactiae in one sample (1/1). Conclusions We found MALDI-TOF-MS to be useful in the rapid identification of Gram-negative rods directly from smear-positive CSF samples, but not of Gram-positive bacteria. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) is a rapid methodology for identification of bacteria that is increasingly used in diagnostic laboratories. This work aimed at evaluating the potential of MALDI-TOF-MS for identification of the main serotypes of Flavobacterium psychrophilum isolated from salmonids, and its discrimination from closely related Flavobacterium spp. A mass spectra library was constructed by analysing 70 F. psychrophilum strains representing the serotypes O1, O2a, O2b and O3, including reference and clinical isolates. Peak mass lists were examined using the Mass-Up software for the detection of potential biomarkers, similarity and cluster analysis. Fourteen species-identifying biomarkers were detected in all the F. psychrophilum isolates tested, moreover, sets of serotype-identifying biomarkers ions were selected. F. psychrophilum -specific biomarkers were identified as ribosomal proteins by matching with protein databases. Furthermore, sequence variation corresponding to amino acid exchanges in several biomarker proteins were tentatively assigned. Closely related Flavobacterium species ( F. flevense , F. succinicans , F. columnare , F. branchiophilum and F. johnsoniae ) could be differentiated from F. psychrophilum by defining species identifying biomarkers and hierarchical cluster analysis. These results demonstrated that MALDI-TOF spectrometry represents a powerful tool for an accurate identification of the fish pathogen F. psychrophilum as well as for epidemiological studies. Biological significance The results obtained in this study demonstrated that MALDI-TOF mass spectrometry represents a powerful tool that can be used by diagnostic laboratories for rapid identification of the fish pathogen Flavobacterium psychrophilum and its differentiation from other Flavobacterium -related species. Analysis of mass peak lists revealed the potential of the MALDI-TOF technique to identify epidemiologically important serotypes affecting salmonid fish. Furthermore, this study revealed the importance of the technique in the early and fast detection of bacterial pathogens, with the subsequent improvement in the health status and animal welfare and food safety in farmed fish. [ABSTRACT FROM AUTHOR]

This study assessed the immobilization of laccase on fibrous polymer-grafted polypropylene chloride film and the removal of three different dyes (i.e., Procion Green H4G, Brilliant Blue G, and Crystal Violet) using immobilized laccase. The polypropylene chloride (PP) film and poly(glycidylmethacrylate)-grafted (PP-g-pGMA) film were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and contact angle studies. The PP-g-pGMA film was used for the direct immobilization of laccase. The amount of immobilized enzyme and the enzyme activity on the film were found to be 1.185 μg/cm 2 and 1.71 × 10 −2 U/cm 2 film, respectively. The retained activity of the immobilized laccase was 72.3%. The optimal conditions for the free and immobilized enzymes and the kinetic parameters were determined. Additionally, the thermal, storage, and operational stabilities of the immobilized laccase were increased compared with those of the free form. Kinetic parameters V max and K m values were determined as 23.4 U/mg protein and 0.38 mmol/L for free enzyme and 17.5 U/mg protein and 0.53 mmol/L for the immobilized laccase, respectively. The removal of the tested dyes with the immobilized laccases was evaluated in batch system and enzyme reactor. The maximum enzymatic removal of tested dyes was achieved at a pH of 5.5 and a temperature of 30 °C. The complete degradation time of the tested dyes was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI–ToF-MS). These results indicate that the immobilized laccase system can be effectively used for the removal of dyes, and it has a great potential for various biotechnological applications. [ABSTRACT FROM AUTHOR]

Bacillus methylotrophicus DCS1 strain was isolated from diesel contaminated soil and screened for its ability to produce biosurfactants; it was found effective for the production of surface active molecules. The structural characterization of the isolated lipopeptides was studied by a variety of analytical techniques. The organic extract of DCS1 lipopeptides was fractionated by silica gel column chromatography (60 Mesh). Fractions containing lipopeptides were collected and identified by tandem mass spectrometry MALDI-TOF-MS and MALDI-TOF MS 2 . The crude biosurfactants contains a mixture of homologous lipopeptides with molecular weights between 1016 and 1556 Da. Mass spectrometry analysis of partially purified lipopeptides revealed that it contains different isoforms belonging to three families: surfactin, iturin and fengycin. To identify lipopeptides isoforms, MALDI-TOF MS 2 was used and ions representing characteristic fragmentations were detected. The mass spectrometry characterization revealed the presence of four variants of surfactin lipopeptides, four variants of pumilacidin that differ according to the β-hydroxy fatty acid chain length as well as the type of amino acid at position 7, five variants of iturin A/mycosubtilin varying in the β-amino fatty acid chain length from C12 to C16, C16 iturin C1, five isoforms of bacillomycin D varying in the β-amino fatty acid chain length from C14 to C18, and six fengycin isoforms that differ according to the length of the β-hydroxy fatty acid side chain as well as the amino acid at position 6. The capacity of B. methylotrohicus DCS1 strain to produce many lipopeptides isoforms belonging to different families and having a structural diversity is a very interesting characteristic that allows them to be used in various fields of biotechnological applications. [ABSTRACT FROM AUTHOR]

It is challenging to apply matrix-assisted laser desorption/ionization−time of flight mass spectrometry (MALDI-TOF MS) with analysis of small molecules efficiently. In this study, we describe the use of SBA-15@APTES@CHCA as a novel silica-containing matrix to aid the analysis of small molecules with MALDI-TOF MS. To synthesize this novel matrix, 3-aminopropyltriethoxysilane (APTES) was used to bond α-cyano-4-hydnoxycinnamic acid (CHCA) and SBA-15 via Si O and three CH 2 alkyl chain. Furthermore, APTES also acts as flexible chains to allow CHCA molecules spreading over the surface of SBA-15, which increase the capacity of matrix to absorb energy and crystallize with sample. This novel matrix is more stable than an ionic macro-complex matrix modified with SBA-15 and can be vacuum dried or air dried easily and stored for a long period as powder. The structure of matrix was characterized by FTIR spectra and SEM. Twelve quinolone antibiotics (danofloxacin, enrofloxacin, ciprofloxacin, difloxacin, enoxacin, gatifloxacin, fleroxacin, lomefloxacin, sparfloxacin, norfloxacin, levofloxacin, pefloxacin, coumaphos, fenobucarb, carbendazim) and three pesticides (carbendazim, coumaphos, bassa) were applied to verify the feasibility of SBA-15@APTES@CHCA as a matrix for small molecules analysis. Several factors which affected the application of this matrix with mass spectrum analysis are discussed. The factors include suspension agent, matrix concentration, matrix/analyte ratio (v: v), sample preparation methods Compared with CHCA, as a matrix, SBA-15@APTES@CHCA displays distinct advantages on analysis of small molecules such as minimal chemical noise and improved analyte signal intensity. It is therefore a promising matrix to be used in MALDI-TOF MS analysis of low molecular weight analytes. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer (EOC) is worldwide the sixth most lethal form of cancer occurring in women. More than one third of ovarian patients have ascites at the time of diagnosis and almost all of them have it when recurrence occurs. Although its effect on tumor cell microenvironment remains poorly understood, its presence is correlated with bad diagnosis. In previous studies, we proposed a novel glycan-based biomarker for the diagnosis of EOC, which showed an improved sensitivity and specificity at any stage of the disease and an improved discrimination between malignant and benign ovarian tumors. In this work, we report for the first time the N-glycome profiles of ascitic fluid from primary serous EOC patients and compare them with the serum N-glycomes of the same patients as well as of healthy controls. N-Glycans were digested from equivalent amount of ascites and serum from 18 EOC patients and from serum of 20 age-matched controls and measured by MALDI-TOF-MS. Ascites N-glycome showed increased antennarity, branching, sialylation and Lewis X motives compared to healthy serum. In addition, a correlation was established between ascites volume and degree of sialylation. Significance Malignant ascitic fluid is the build-up of large volumes of fluid in the peritoneal cavity secondary to cancer. At least one-third of ovarian cancer patients develop ascites, a generally voluminous fluid containing cells of tumor origin, in the course of cancer and almost all when recurrence occurs. The proteome of ascites is known to be as complex as that of serum and contains high amount of proteins shed from inflammatory cells as well as from tumor cells. Although many attempts have been made to provide molecular insight into the proteomic and peptidomic content of malignant ascites, no data about the N-glycome of the ascitic fluid fraction from cancer patients has been reported to date. In this study, the N-glycosylation profile of ascites from 20 patients suffering from epithelial ovarian cancer (EOC) was analyzed by MALDI-TOF-mass spectrometry and compared to the pathologically modified N-glycan pattern obtained from serum of the same patients as well as to the pattern of serum from healthy individuals. Significant quantitative differences were observed in the ascites of EOC patients when compared to the serum of healthy subjects. The glycome of ascites shows typical features of inflammatory conditions, what was also found in the serum of patients suffering from EOC when compared to healthy serum. In addition, a correlation was established between ascites volume and degree of sialylation, showing that the high-volume ascites contains a higher amount of sialylated structures than the low-volume ascites. [ABSTRACT FROM AUTHOR]

Cellulose ethers used as adhesives in heritage conservation treatments have been successfully identified by means of MALDI-TOF-MS, a technique non-previously applied for this purpose in cultural assets. This is of relevant importance for long-term conservation, as discrimination among the diverse types of cellulose ethers that may have been applied to an asset during conservation treatments is essential in order to guarantee stability of artworks. The proposed method also allows discrimination among these adhesives spread on paper-based artworks, where cellulose ethers have been extensively utilized for many years, overcoming interferences usually occurred due to the cellulosic nature of both adhesive and support. Successful results have been obtained from mock-ups and small samples of paper-based original artworks with usual low concentrations of adhesive. FTIR and NMR have been used as complementary analytical techniques. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been proven as a useful and advanced technique in the identification of polymers and proteins. However, MALDI-TOF-MS still has the unavoidable drawback of self-signal interference with traditional organic matrices, which could suppress and overlap with the analyte signals in the low-mass region. In this work, MIL-101(Cr), a kind of metal-organic frameworks which possess high molecular weight, π-conjugated 3-D structure, coordinately unsaturated chromium sites (CUS) and strong absorption in the UV range, was employed to replace traditional organic matrices, and it was found that MIL-101(Cr) can dramatically eliminate the background peaks, showing high signal-to-noise level in the analysis of small molecules. As proof-of-concept, quercetin, daidzein, genistein and naringenin, members of flavonol family which widely exists in food and natural products, were successfully determined by utilizing MIL-101(Cr) as the surface-assisted matrix, and the detection of quercetin was sensitive with good salt tolerance and reproducibility. Under optimal conditions, the mass peak intensity exhibited good linear relationships in the range from 0.25 µg/mL-7.00 µg/mL for quercetin ( R 2 =0.996) with detection limit 2.11 ng/mL (3σ/k), making the identification of quercetin in sophora japonica successfully. With this strategy we have demonstrated the potentiality of MIL-101(Cr) nanomaterials as MALDI-MS matrix for the detection of small molecules. [ABSTRACT FROM AUTHOR]

The genotype (G), environment (E), and the relationship between G and E on soybean seed anti-nutritional factors (ANF’s) were examined under three different agro-climatic conditions. The field trials were conducted at Maryland, South Carolina and South Dakota using nine region specific genotypes. At each location, the nine genotypes were grown with two planting/sowing dates. Differentially expressed protein spots from the two-dimensional gel electrophoresis were analyzed using mass spectrometry. Seven ANF’s corresponding to soybean agglutinin and Kunitz trypsin inhibitor were identified based on the statistical significance levels at p < 0.005. The G and E conditions (planting/sowing season) influences the ANF’s content. This initial study suggests that early sowing reduces the total ANF’s content irrespective of genotypes and their growing locations. [ABSTRACT FROM AUTHOR]

The existence form and structure properties of mobile phase (MP) in low-rank coals (LRCs) can significantly influence the initial stage of thermal conversion. In the present work, three Chinese LRCs, namely, Shenfu, Zhundong and Hongshaquan, were extracted with tetrahydrofuran using the microwave-assisted heating. The tetrahydrofuran-microwave-extracted (TME) portion as the representative of MP was further separated to four fractions defined as oil, resin, asphaltene and preasphaltene, respectively. Diffuse reflectance Fourier transform-infrared spectroscopy (DRIFT), gas chromatography/mass spectrometer (GC/MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) and X-ray photoelectron spectroscopy (XPS) were used to comprehensively investigate the molecular characteristics of the derived materials. The results indicated that the studied TME portions were mainly consisted of asphaltenes and rich in highly branched aliphatic hydrocarbons due to the relatively low CH 2 /CH 3 and H ar /H al ratios. Para -alkyl substituted aromatic structures with 1–2 rings were the main aromatic structures in the TME portions. C O bonds were the main oxygen-containing structures in the TME portions and could be more likely seen in aliphatic compounds. Combining the MALDI-TOF-MS and DRIFT analyses, the ratio of aliphatic side chains and aromatic hydrogens (3000–2800 cm −1 /900–700 cm −1 , I 2 ) derived from IR spectra seemed to be a suitable parameter for assessing the average molecular weight (AMW) of the specific fraction in TME portion of LRCs when the ratio of C O/C O was at very low level. The results made a further explanation for the detailed chemical structure of mobile phase in coal and could be helpful for studying the formation mechanism of volatiles during pyrolysis process. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been commonly used for the determination of macromolecules owing to the high sensitivity and convenience. However, the direct analysis of small molecule with MALDI-TOF-MS remains a challenge due to the matrix interference in the low mass region. In this paper, metal-organic frameworks (MOFs: MOF-5, MOF-235, Cu-btc MOFs and Uio-66(Zr)-2OH) were successfully used as both adsorbent and significant matrix of MALDI-TOF-MS method for the analysis of small molecule pollutants. Benzo( a )pyrene (BaP) and perfluorinated compounds (PFCs) were chosen as model analytes to investigate the matrix function of MOFs with LDI measurement in positive and negative reflection mode, respectively. As a result, MOFs exhibit high laser desorption/ionization efficiency and function well in both positive-ion and negative-ion modes. Compared to the MS spectra with conventional matrices, MS spectra obtained on MOFs matrix are only featured by deprotonated molecule ion peaks without background interference. Furthermore, MOFs, with large surface area and π-π stacking structure, can also enrich trace amounts of small molecule pollutants from solution efficiently and quickly. According to these, we developed a simple method in which MOFs-solid-phase extraction (SPE) enrichment was combined with MALDI-TOF-MS to realize the direct analysis of perfluorooctanesulfonate (PFOS) in real water samples. [ABSTRACT FROM AUTHOR]

The ability of phosphonium cations to act as intracellular transport vectors is well-established. Phosphonioalkylthiosulfate zwitterions, and ω-thioacetylalkylphosphonium salts, which act as 'masked thiolate' ligands, are useful precursors for the formation of phosphonium-functionalised gold nanoparticles, enabling the nanoparticles to be transported into cells for diagnostic and therapeutic purposes. In this study we have completed cytotoxicity studies of ω-thioacetylpropylphosphonium salts derived from triphenylphosphine and tri(4-fluorophenyl)phosphine, which show that the compounds are only toxic towards PC3 prostate cancer cells at high concentrations and at prolonged incubation periods and display IC 50 values of 67 μM and 252 μM respectively, significantly higher than those of other phosphonium salts. MALDI-TOF-MS has been used to investigate the uptake of the compounds by PC3 cells and to quantify detectable levels of the compounds inside the cells. The structures of ω-thioacetylpropyl(tri-4-fluorophenyl) phosphonium bromide and the corresponding tri(4-fluorophenyl)phosphoniopropylthiosulfate zwitterion have been investigated by single crystal X-ray crystallography. The results show that molecules of the zwitterion are held together through an extensive array of electrostatic and non-covalent interactions. The unit cell of ω-thioacetylpropyl(tri-4-fluorophenyl)phosphonium bromide contains eight cations together with eight bromide anions and two waters of crystallisation, all held together through a complex network of hydrogen bonds. The differences in the molecular packing of the two compounds may account for the lower solubility of the zwitterion in aqueous solutions, compared with that of the phosphonium salt. [ABSTRACT FROM AUTHOR]

[Display omitted] • Effect of S. baltica on texture and flavour of golden pomfret were investigated. • S. baltica decreased fish hardness, and the storage and loss moduli of proteins. • MHC, MyBP-C and actin were degraded by S. baltica. • S. baltica produced dimethyl sulfide, 2-methyl-butanal and 3-methyl-butanal. Shewanella baltica has a high spoilage ability to decompose nutrients in fish. To investigate the role of S. baltica in fish protein and flavour during spoilage, the texture, rheology, protein patterns and volatile compounds of golden pomfret inoculated with S. baltica during 10-day storage were tested. During storage, S. baltica reduced the hardness of fish sticks by 29.73–49.24 %. Compared to the control (G 0 ′: 20.27 ± 2.15 kPa), inoculated samples showed lower moduli (G 0 ′: 16.71 ± 0.82–17.50 ± 1.80 kPa). Their myosin heavy chains, myosin-binding protein C and actin were decomposed into smaller proteins, which was validated by the lower intensities of molecules with M w 160–176 kDa. Furthermore, S. baltica generated volatile spoilage markers, including dimethyl sulfide, 2-methyl-butanal and 3-methyl-butanal. This study reveals the mechanism of fish texture and flavour changes induced by S. baltica , and provides insights into controlling bacterial spoilage of seafood. [ABSTRACT FROM AUTHOR]

[Display omitted] • Immunohistochemistry with magnetic core nanoparticles to isolate viruses. • The use of MALDI-MS for rapid virus detection is explained in detail. • The use of ESI-MS/MS to pinpoint host-patient crosstalk is explained in detail. • The absolute quantitative MS is explained for large-scale protein quantitation. The capabilities of bioanalytical mass spectrometry to (i) detect and differentiate viruses at the peptide level whilst maintaining high sample throughput and (ii) to provide diagnosis and prognosis for infected patients are presented as a tutorial in this work to aid analytical chemists and physicians to gain insights into the possibilities offered by current high-resolution mass spectrometry technology and bioinformatics. From (i) sampling to sample treatment; (ii) Matrix-Assisted Laser Desorption Ionization- to Electrospray Ionization -based mass spectrometry; and (iii) from clustering to peptide sequencing; a detailed step-by-step guide is provided and exemplified using SARS-CoV-2 Spike Y839 variant and the variant of concern SARS-CoV-2 Alpha (B.1.1.7 lineage), Influenza B, and Influenza A subtypes AH1N1pdm09 and AH3N2. [ABSTRACT FROM AUTHOR]

• MALDI-TOFD- MS directly confirmed β -casein-4-hydroxybenzoic acid covalent complexes. • A 3D structure of covalently bound β -casein-4-hydroxybenzoic acid was developed. • Molecular dynamics simulations indicated changes to solvent accessibility. • Altered hydrogen bonding on complexation led to decreased protein structural stability. Previous work has suggested the ability of 4-hydroxybenzoic acid (4HBA) to bind to β - casein following ultra high temperature-like processing (UHT) in model aqueous systems. The present work confirmed directly, using MALDI-TOF-MS, the presence of covalently bound 4HBA following UHT-like treatment. In subsequent molecular dynamics simulations, the 3D structure of the β - casein molecule was modified so that the meta -C of 4HBA ring and the side chain amino group of lys32 were linked covalently. Such simulations further indicated that the covalent addition of the phenolic compound had impacted the protein density and solvent accessibility. Hydrogen bond analysis between lys32 and the remainder of the protein structure revealed that the covalent complexation supported the formation of additional hydrogen bonds. These increased from potentially 9, in the single protein molecule, to 51 in the complex with 4HBA. However, the persistence of hydrogen bonds was reduced, leading overall to decreased stability and increased protein flexibility. [ABSTRACT FROM AUTHOR]

• MALDI-TOF-MS with chemometrics is described for classification of quinoa grains. • Mass spectra for protein extracts from 4 commercial quinoa grains were obtained. • MALDIquant allowed reliable detection of 47 quinoa grain proteins. • 30 of the detected quinoa grain proteins were tentatively identified. • Quinoa grains were classified by PLS-DA based on their differential protein composition. Quinoa is an Andean grain that is attracting attention worldwide as a high-quality protein-rich food. Nowadays, quinoa foodstuffs are susceptible to adulteration with cheaper cereals. Therefore, there is a need to develop novel methodologies for protein characterization of quinoa. Here, we first developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method to obtain characteristic mass spectra of protein extracts from 4 different commercial quinoa grains, which group different varieties marketed as black, red, white (from Peru) and royal (white from Bolivia). Then, data preprocessing and peak detection with MALDIquant allowed detecting 47 proteins (being 30 tentatively identified), the intensities of which were considered as fingerprints for multivariate data analysis. Finally, classification by partial least squares-discriminant analysis (PLS-DA) was excellent, and 34 out of the 47 proteins were critical for differentiation, confirming the potential of the methodology to obtain a reliable classification of quinoa grains based on protein fingerprinting. [ABSTRACT FROM AUTHOR]

An extraction procedure using Tris-HCl buffer solution was employed in order to extract water-soluble proteins from seed samples of oat, wheat and soybean. Initially, the total protein concentration was determined by the Bradford method in each solution, after the extraction procedure. The soybean sample showed a higher concentration of total protein compared to the others. The protein extracts obtained were separated by reverse-phase chromatography (RP-HPLC-UV). The protein fractions were collected and analyzed by graphite furnace atomic absorption spectrometry (GFAAS) and matrix-assisted laser desorption/ionization (MALDI-TOF-MS) for determination of Cu, Fe, Mn and Zn and identification of proteins, respectively. The combination of techniques such as RP-HPLC-UV, GFAAS and MALDI-TOF-MS allowed the identification of several proteins bound to metals present in the seed samples. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) becomes the method of choice for the rapid identification of microorganisms (i.e. protein biotyping). Although bacterial identification is already quite advanced, biotyping of other microbes including yeasts and fungi are still under development. In this context, lipids (e.g. membrane phospholipids) represent a very important group of molecules with essential functions for cell survival and adaptation to specific environments and habitats of the microorganisms. Therefore, lipids show the potential to serve as additional molecular parameters to be used for biotyping purposes. In this paper we present a molecular characterisation of yeasts and filamentous fungi based on the analysis of lipid composition by MALDI-MS (i.e. MALDI lipid phenotyping ). Using a combination of Principal Component Analysis (PCA) and Hierarchical Clustering we could demonstrate that this approach allowed a classification and differentiation of several groups of yeasts (e.g. Saccharomyces ) and filamentous fungi (e.g. Aspergillus , Penicillium , Trichoderma ) at the species/strain level. By analysing the MALDI lipid profiles we were able to differentiate 26 closely related yeast strains, for which discrimination via genotypic methods like AFLP in this case are relatively more elaborate. Moreover, employing statistical analysis we could identify those lipid parameters (e.g. PCs and LPCs), which were responsible for the differentiation of the strains, thus providing insights into the molecular basis of our results. In summary, MALDI lipid phenotyping represents a suitable method for fungal characterization and shows the potential to be used as companion tool to genotyping and/or protein biotyping for the characterization and identification of yeasts and fungi in diverse areas (e.g. environmental, pharmaceutical, clinical applications, etc.). [ABSTRACT FROM AUTHOR]

Efficient and specific enrichment of low-abundant glycopeptides from complex biological samples is essential to glycoproteomics analysis. Herein, novel magnetic zwitterionic hydrophilic nanoparticles based on thiol-ene click chemistry were synthesized. The functionalized magnetic nanoparticles exhibited superior performance in glycopeptide enrichment of HRP tryptic digest, demonstrating low detection limit (0.04 ng/μL), high selectivity (a mixture of HRP and BSA at the mass ratio of 1:50) and reproducibility (5 repeating cycles). In addition, the material was successfully applied to glycopeptide enrichment from human serum. The outstanding results indicate the potential of the method in the development of glycoproteomics analysis in real biological samples. [ABSTRACT FROM AUTHOR]