Matrix-assisted Laser Desorption/ionization Time-of-flight Mass Spectra Research Articles

Synthesis and end-group control of soluble, high-molecular-weight hyperbranched poly(phenylene thienylene)s by non-stoichiometric A2 + B3 Suzuki-Miyaura polycondensation without gelation

Non-stoichiometric Suzuki-Miyaura polycondensation of tribromo monomer 1, containing a thienylene group, with diboronic acid (ester) monomer 7 in the presence of tBuPPd catalyst, which has a propensity for intramolecular catalyst transfer on a π-electron face, was investigated. We anticipated that the presence of thienylene groups in the obtained hyperbranched polymer would increase the detection sensitivity in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and enable the analysis of the polymer end groups up to the high-molecular-weight region. We first conducted Suzuki-Miyaura reaction of several kinds of 1 with phenylboronic acid 2 in the presence of tBuPPd precatalyst 3. The reaction of tris(5-bromo-4-hexylthien-2-yl)benzene 1c with 2 afforded exclusively tri-substituted product 4c, indicating that the tBuPPd catalyst undergoes intramolecular catalyst transfer on 1c after the first and second substitutions of 1c with 2. The polycondensation of equimolar 1c and pinacol phenylenediboronate 7a (reaction site ratio of Br:BPin = 1.5:1.0 (BPin = pinacol boronate)) was then carried out in the presence of 3 for 3 h, affording hyperbranched polymer with Mn = 37500 without gelation. The product was purified by precipitation. The degree of polymerization (DP) was calculated from the absolute molecular weight, determined by multi-angle light scattering (MALLS) analysis, to be 200, which is a far higher than would be predicted from the Flory-Stockmayer theory at the critical gel point (DP = 6.6). Furthermore, the MALDI-TOF mass spectra showed two series of peaks due to polymer with boronate ends and boronate ends along with one cyclic unit up to m/z of about 7500, as we had expected. The absence of peaks due to polymer with Br ends indicates that the degree of branching (DB) of this hyperbranched polymer is 100 %.

Automatic antibiotic resistance prediction in Klebsiella pneumoniae based on MALDI-TOF mass spectra

Matrix-Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) Mass Spectrometry (MS) is a reference method for microbial identification and it can be used to predict Antibiotic Resistance (AR) when combined with artificial intelligence methods. However, current solutions need time-costly preprocessing steps, are difficult to reproduce due to hyperparameter tuning, are hardly interpretable, and do not pay attention to epidemiological differences inherent to data coming from different centres, which can be critical.We propose using a multi-view heterogeneous Bayesian model (KSSHIBA) for the prediction of AR using MALDI-TOF MS data together with their epidemiological differences. KSSHIBA is the first model that removes the ad-hoc preprocessing steps that work with raw MALDI-TOF data. In addition, due to its Bayesian probabilistic nature, it does not require hyperparameter tuning, provides interpretable results, and allows exploiting local epidemiological differences between data sources. To test the proposal, we used data from 402 Klebsiella pneumoniae isolates coming from two different domains and 20 different hospitals located in Spain and Portugal. KSSHIBA outperforms current state-of-the-art approaches in antibiotic susceptibility prediction, obtaining a 0.78 AUC score in Wild Type classification and a 0.90 AUC score in Extended-Spectrum Beta-Lactamases (ESBL)+Carbapenemases (CP)-producers. The proposal consistently removes the need for ad-hoc preprocessing by working with raw MALDI-TOF data, which, in turn, reduces the time needed to obtain the results of the resistance mechanism in microbiological laboratories. The proposed model implementation as well as both data domains are publicly available.

Direct antimicrobial resistance prediction from clinical MALDI-TOF mass spectra using machine learning.

Early use of effective antimicrobial treatments is critical for the outcome of infections and the prevention of treatment resistance. Antimicrobial resistance testing enables the selection of optimal antibiotic treatments, but current culture-based techniques can take up to 72 hours to generate results. We have developed a novel machine learning approach to predict antimicrobial resistance directly from matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectra profiles of clinical isolates. We trained calibrated classifiers on a newly created publicly available database of mass spectra profiles from the clinically most relevant isolates with linked antimicrobial susceptibility phenotypes. This dataset combines more than 300,000 mass spectra with more than 750,000 antimicrobial resistance phenotypes from four medical institutions. Validation on a panel of clinically important pathogens, including Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, resulting in areas under the receiver operating characteristic curve of 0.80, 0.74 and 0.74, respectively, demonstrated the potential of using machine learning to substantially accelerate antimicrobial resistance determination and change of clinical management. Furthermore, a retrospective clinical case study of 63 patients found that implementing this approach would have changed the clinical treatment in nine cases, which would have been beneficial in eight cases (89%). MALDI-TOF mass spectra-based machine learning may thus be an important new tool for treatment optimization and antibiotic stewardship.

Topological and kernel-based microbial phenotype prediction from MALDI-TOF mass spectra.

MotivationMicrobial species identification based on matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has become a standard tool in clinical microbiology. The resulting MALDI-TOF mass spectra also harbour the potential to deliver prediction results for other phenotypes, such as antibiotic resistance. However, the development of machine learning algorithms specifically tailored to MALDI-TOF MS-based phenotype prediction is still in its infancy. Moreover, current spectral pre-processing typically involves a parameter-heavy chain of operations without analyzing their influence on the prediction results. In addition, classification algorithms lack quantification of uncertainty, which is indispensable for predictions potentially influencing patient treatment.ResultsWe present a novel prediction method for antimicrobial resistance based on MALDI-TOF mass spectra. First, we compare the complex conventional pre-processing to a new approach that exploits topological information and requires only a single parameter, namely the number of peaks of a spectrum to keep. Second, we introduce PIKE, the peak information kernel, a similarity measure specifically tailored to MALDI-TOF mass spectra which, combined with a Gaussian process classifier, provides well-calibrated uncertainty estimates about predictions. We demonstrate the utility of our approach by predicting antibiotic resistance of three clinically highly relevant bacterial species. Our method consistently outperforms competitor approaches, while demonstrating improved performance and security by rejecting out-of-distribution samples, such as bacterial species that are not represented in the training data. Ultimately, our method could contribute to an earlier and precise antimicrobial treatment in clinical patient care.Availability and implementationWe make our code publicly available as an easy-to-use Python package under https://github.com/BorgwardtLab/maldi_PIKE.

Matrix-assisted laser desorption/ionization behavior of neat linear and cyclic poly(L-lactide)s and their blends.

RationaleNumerous new tin catalysts that enable the synthesis of cyclic polylactides with broad variation in their molecular mass were recently developed. The abundance of cyclics in matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectra is, however, frequently reported to greatly exceed that of linears. Thus, the MALDI ionization behavior of various end‐capped linear poly(L‐lactide)s and one cyclic poly(L‐lactide) was investigated and compared.MethodsNeat compounds and various blends of cyclic and linear species were prepared and studied under identical conditions with regard to sample preparation and instrumental condition, except for the laser power. For this purpose, two different MALDI‐TOF mass spectrometers were applied.ResultsOur results reveal that cyclics indeed show a slightly better ionization in MALDI, although their ionization as a neat compound seems to be less effective than that of linear polylactides. The ionization of most linear polylactides investigated does not depend on the end group structure. However, linear polylactides containing 12‐bromododecyl end groups reveal an unexpected saturation effect that is not caused by fragmentation of the polymer or the end group, or by electronic saturation of the detector digitizer. Furthermore, polylactides with a 2‐bromoethyl end group did not show such a saturation effect.ConclusionsAn overestimation of cyclic species in MALDI‐TOF mass spectra of poly(L‐lactide)s must be considered, but the commonly assumed peak suppression of linear polymers in mixtures of both structures can be excluded.

MALDI-TOF Mass Spectrometry Based on Parylene-Matrix Chip for the Analysis of Lysophosphatidylcholine in Sepsis Patient Sera.

In this work, medical diagnosis of sepsis was conducted via quantitative analysis of lysophosphatidylcholine 16:0 (LPC 16:0) by using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry based on a parylene-matrix chip. In the first step, specific mass peaks for the diagnosis of sepsis were searched by comparing MALDI-TOF mass spectra of sepsis patient sera with healthy controls and pneumonia patient sera. Two mass peaks at m/z = 496.3 and 518.3 were chosen as those that are specifically different for sepsis sera to compare with healthy controls and pneumonia patient sera. These mass peaks were identified to be protonated and sodium adducts of LPC 16:0 by using tandem mass spectra (MS2 and MS3) of purely synthesized LPC 16:0 and extracted LPC 16:0 from a healthy control and a sepsis patient. In the next step, a standard curve for LPC 16:0 for the quantitative analysis of LPC 16:0 with MALDI-TOF MS based on the parylene-matrix chip was prepared, and the statistical correlation to the LC-MS analysis results was demonstrated by using the Bland-Altman test and Passing-Bablok regression. Finally, MALDI-TOF MS based on the parylene-matrix chip was used for the quantification of LPC 16:0 with sera from patients with severe sepsis and septic shock (n = 143), pneumonia patients (n = 12), and healthy sera (n = 31). The sensitivity and the selectivity of medical diagnosis of sepsis was estimated to be 97.9% and 95.5% by using MALDI-TOF MS based on the parylene-matrix chip, respectively.

Bacterial iturins mediate biocontrol activity of Bacillus sp. against postharvest pear fruit‐rotting fungi

AbstractA potential antagonist, Bacillus sp. LYLB4 isolated from pear fruits, was tested for its antifungal activity against postharvest pear pathogens. LYLB4 had a remarkable antifungal effect on Botryosphaeria dothidea. Although it showed a weak inhibition effect on the growth of Rhizopus stolonifer on potato dextrose agar (PDA) plates, LYLB4 almost completely destroyed R. stolonifer during direct contact in potato dextrose broth (PDB). LYLB4 treatment was able to significantly reduce disease incidence (by 68.9% and 100%, respectively) and lesion diameter (by 68.7% and 100%, respectively) of ring rot caused by B. dothidea and soft rot caused by R. stolonifer in pears. LYLB4 also suppressed several other phytopathogens in vitro, suggesting a broad‐spectrum antagonistic activity against fungal pathogens. 16S rRNA and gyrA sequence analysis indicated that LYLB4 is closely related to B. velezensis. Genome mining indicated that LYLB4 had 11 secondary metabolites encoding clusters, but that the surfactin and fengycin gene clusters may not be functional because of a large deletion. Matrix‐assisted laser desorption ionization‐time of flight mass spectra (MALDI‐TOF‐MS) demonstrated that iturins were the major lipopeptides, and that C16/C17 Bacillomycin D synthesis was stimulated when LYLB4 was co‐cultured with B. dothidea compared to the control. Overall, these results demonstrate that the main biocontrol mechanism adopted by LYLB4 could be through the production of toxic metabolites and direct contact with pathogens.

Whole animal matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of ticks - Are spectra of Ixodes ricinus nymphs influenced by environmental, spatial, and temporal factors?

In the recent years matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has become a useful tool to characterize arthropod species and their different stages of development. It was reported for sand flies and mosquitoes at immature stages and also assumed for ticks that geographic location can have a subtle influence on MALDI-TOF mass spectra which allows the discrimination of animals with specific local variations of the MALDI-TOF MS phenotype. It is so far uncertain, however, if these mass-spectrometric differences are based on genetic variation or on spectral features which depend on environmental or temporal features. The aim of this study was to analyze the influence of the geographic location, environmental factors and the season of the year on the MALDI-TOF mass spectra of Ixodes (I.) ricinus nymphs and if spectral variation would allow to draw conclusions with respect to the tick’s provenience or conditions that influence the tick life cycle. Application of multivariate statistical models on spectra of ticks collected in different seasons and different habitats and locations within Germany showed that the impact of the location seemed to be small while season and habitat seemed to have stronger impact on the MALDI-TOF mass spectra. Possibilities and limitations of MALDI-TOF mass spectra to draw conclusions on the tick life cycle are discussed.

Rapid Microbial Identification and Antibiotic Resistance Detection by Mass Spectrometric Analysis of Membrane Lipids.

Infectious diseases have a substantial global health impact. Clinicians need rapid and accurate diagnoses of infections to direct patient treatment and improve antibiotic stewardship. Current technologies employed in routine diagnostics are based on bacterial culture followed by morphological trait differentiation and biochemical testing, which can be time-consuming and labor-intensive. With advances in mass spectrometry (MS) for clinical diagnostics, the U.S. Food and Drug Administration has approved two microbial identification platforms based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis of microbial proteins. We recently reported a novel and complementary approach by comparing MALDI-TOF mass spectra of microbial membrane lipid fingerprints to identify ESKAPE pathogens. However, this lipid-based approach used a sample preparation method that required more than a working day from sample collection to identification. Here, we report a new method that extracts lipids efficiently and rapidly from microbial membranes using an aqueous sodium acetate (SA) buffer that can be used to identify clinically relevant Gram-positive and -negative pathogens and fungal species in less than an hour. The SA method also has the ability to differentiate antibiotic-susceptible and antibiotic-resistant strains, directly identify microbes from biological specimens, and detect multiple pathogens in a mixed sample. These results should have positive implications for the manner in which bacteria and fungi are identified in general hospital settings and intensive care units.

Rapid Detection of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Based on Matrix-Assisted Laser Desorption Ionization Time-of-Flight: Using a Machine Learning Approach and Unbiased Validation.

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) is an emerging superbug with implicit drug resistance to vancomycin. Detecting hVISA can guide the correct administration of antibiotics. However, hVISA cannot be detected in most clinical microbiology laboratories because the required diagnostic tools are either expensive, time consuming, or labor intensive. By contrast, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) is a cost-effective and rapid tool that has potential for providing antibiotics resistance information. To analyze complex MALDI-TOF mass spectra, machine learning (ML) algorithms can be used to generate robust hVISA detection models. In this study, MALDI-TOF mass spectra were obtained from 35 hVISA/vancomycin-intermediate S. aureus (VISA) and 90 vancomycin-susceptible S. aureus isolates. The vancomycin susceptibility of the isolates was determined using an Etest and modified population analysis profile–area under the curve. ML algorithms, namely a decision tree, k-nearest neighbors, random forest, and a support vector machine (SVM), were trained and validated using nested cross-validation to provide unbiased validation results. The area under the curve of the models ranged from 0.67 to 0.79, and the SVM-derived model outperformed those of the other algorithms. The peaks at m/z 1132, 2895, 3176, and 6591 were noted as informative peaks for detecting hVISA/VISA. We demonstrated that hVISA/VISA could be detected by analyzing MALDI-TOF mass spectra using ML. Moreover, the results are particularly robust due to a strict validation method. The ML models in this study can provide rapid and accurate reports regarding hVISA/VISA and thus guide the correct administration of antibiotics in treatment of S. aureus infection.

Effect of cytochrome P450 2C19 genotypic polymorphism on treatment efficacy for gastroesophageal reflux disease by rabeprazole

Objective To investigate whether the effect of rabeprazole in treating gastroesophageal reflux disease is related to CYP2C19 gene polymorphisms. Methods 278 patients with gastroesophageal reflux disease confirmed by endoscopy and proton-pump inhibitor testing were enrolled in this study, including non erosive reflux disease (NERD) in 122 cases, the reflux esophagitis (RE) in 98 cases and Barrett esophagus in 58 cases.They were treated with rabeprazole for 8 weeks.GerdQ scores before and after the treatment were completed, endoscopy was performed again in patients with RE after treatment.The blood CYP2C19 genotyping was detected by matrix assisted laser desorption ionization time of flight mass spectrum(MALDI-TOF-MS). Results According to the genotype of CYP2C19, they were divided into extensive metabolizers, intermediate metabolizers and poor metabolizers, accounted for 39.57%, 42.45% and 17.98%, respectively.There was no significant difference in GerdQ scores of three groups before treatment, and also had no significant difference after 8 weeks treatment, but in each subgroup GerdQ scores after treatment was decreased significantly than before treatment.The total effective rate of 98 patients with RE by endoscopy was 86.73%, but there was no significant difference in total effective rate of the three groups after treatment. Conclusion Rabeprazole is effective in the treatment of gastroesophageal reflux disease.Moreover, rabeprazole is less affected by CYP2C19 genotype and therefore its curative effect is more stable. Key words: Gtochrome P-450 enzyme system; Gastroesophageal reflux; Rabeprazole

A Preliminary Investigation Into the Characterization of Asphaltenes Extracted From an Oil Sand and Two Vacuum Residues From Petroleum Refining Using Nuclear Magnetic Resonance, DEPT, and MALDI-TOF

This paper reports the findings of an investigation into the molecular structures and properties of three asphaltene samples, namely, an asphaltene sample extracted from Buton Oil Sand (Indonesia), and two asphaltene samples extracted from vacuum residues from Liaohe Refinery (China) and Vene Refinery (Venezuela), respectively. The average molecular structural parameters, including the average polycyclic aromatic hydrocarbon (PAH) size, average side chain length, and average molecular weight (AMW), of the three asphaltene samples were estimated using data from nuclear magnetic resonance (NMR) in combination with distortionless enhancement by polarization transfer (DEPT), and then compared against each other. The molecular weight distributions (MWDs) of the three asphaltene samples were measured using a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The results indicated that the island molecular architecture predominated in all three asphaltenes and the average polycyclic aromatic hydrocarbon size was found to be six rings. The average molecular weight of the Buton asphaltene sample was found to be ca. 800 Da while those of the two petroleum asphaltene samples were approximately 600 Da. In comparison, the Buton asphaltene sample contained a much higher level of oxygen and sulfur, but a lower aromaticity than those of the two petroleum asphaltene samples. The use of liquid NMR in combination with DEPT was shown to provide an effective method for characterization and estimation of the molecular structures of asphaltenes, supported by MALDI-TOF mass spectra.

A Green Platform for Preparation of the Well-Defined Polyacrylonitrile: 60Co γ-ray Irradiation-Initiated RAFT Polymerization at Room Temperature.

60Co γ-ray irradiation-initiated reversible addition–fragmentation chain transfer (RAFT) polymerization at room temperature with 2-cyanoprop-2-yl 1-dithionaphthalate (CPDN) as the chain transfer agent was first applied to acrylonitrile (AN) polymerization, providing a “green” platform for preparing polyacrylonitrile (PAN)-based carbon fibers using an environment-friendly energy source. Various effects of dose rate, molar ratio of the monomer to the chain transfer agent, monomer concentration and reaction time on the AN polymerization behaviors were performed to improve the controllability of molecular the weight and molecular weight distribution of the obtained PAN. The feature of the controlled polymerization was proven by the first-order kinetics, linear increase of the molecular weight with the monomer conversion and a successful chain-extension experiment. The molecular weight and molecular weight distribution of PAN were characterized by size exclusion chromatography (SEC). 1H NMR and Matrix assisted laser desorption ionization/time of flight mass spectra (MALDI-TOF-MS) confirmed the chain-end functionality of PAN, which also was supported by the successful chain-extension experiments of original PANs with acrylonitrile and styrene as the second monomers respectively.

An evaluation of the impact of urban air pollution on paint dosimeters by tracking changes in the lipid MALDI-TOF mass spectra profile

We evaluated the impact of urban air pollution on egg yolk tempera paint dosimeters (binary mixture samples made with historic artist´s blue, red and white pigments) by tracking changes over time in their lipid matrix-assisted laser desorption ionization time-of-flight mass spectra (MALDI-TOF-MS) profiles. We studied triacylglycerols (TGs), phospholipids (PLs) and their oxidation by-products from paint dosimeters that had been exposed outdoors for six months to the polluted atmosphere in the city center of Granada (Spain). Four types of chickens’ eggs were also analyzed to find out whether their lipid mass spectra (lipid fingerprints) varied significantly. The ultimate goal of this research is to provide a precise analytical protocol to show whether the changes in the egg yolk identified in paint dosimeters are due to pigment-binder interactions. The Bligh-Dyer (BD) method was optimized for the extraction of the lipids. This innovative procedure included a washing-step prior to the mass spectrometric analysis, which proved crucial for obtaining higher quality lipid fingerprints. A novel interpretation of the results is proposed by applying the BD method, which suggests that transesterification processes occurred in the lipid fractions that were catalyzed by the pigments in the paint dosimeters. In blank dosimeters specific ions produced by oxidative cleavage of PLs and/or TGs may be used as markers of the presence of egg yolk binders. The composition and structure of the specific lipid compounds are also tentatively proposed. In aged dosimeters the intact content of the TGs and PLs decreased; however, we propose that short-chain oxidative products arising from TGs and PLs are present in all the samples, except for the white lead based dosimeter. We end with a new explanation as to why this dosimeter behaves differently from the others.

Analysis of Purified Oligomeric Proanthocyanidins from Larix gmelinii Bark and the Study of Physiological Activity of the Purified Product

A simple and efficient method for the purification of oligomeric proanthocyanidins (LOPC) from degreased Larix gmelinii bark was developed. The purity of LOPC was increased from 51.7% to 92.2%, and the cumulative recovery rate was 97.0%. The reversed phase high performance liquid chromatography-mass spectrum (RHPLC-MS) analysis indicated that the percentage contents of catechin (CA), epicatechin (EC), and procyanidin B1 (PB1) in purified LOPC (P-LOPC) were 5.05%, 2.02%, and 0.71%, respectively. The percentage contents of catechin and procyanidin B1 were noticeably higher than those obtained from grape seed extract (2.77% and 0.61%). The average degree of polymerization of P-LOPC was found to be 2.66. The matrix-assisted laser desorption ionization-time of flight/mass spectrum (MALDI-TOF/MS) analysis demonstrated that the dimer was the major component of purified LOPC and the distribution range was from dimer (m/z 713) to decamer (m/z 3016.6). The IC50 values of P-LOPC against DPPH• (1,1-diphenyl-2-picryl-hydrazyl), OH• (hydroxy radical), and ABTS+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) were 76.4±3.82, 7.92±0.40, and 2.4±0.12. Purified LOPC exhibited more excellent physiological activity than VC (vitamin C), TBHQ (tert-butyl hydroquinone), and pine bark extract.

MALDI-TOF typing highlights geographical and fluconazole resistance clusters in Candida glabrata.

Utilizing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra for Candida glabrata typing would be a cost-effective and easy-to-use alternative to classical DNA-based typing methods. This study aimed to use MALDI-TOF for the typing of C. glabrata clinical isolates from various geographical origins and test its capacity to differentiate between fluconazole-sensitive and -resistant strains.Both microsatellite length polymorphism (MLP) and MALDI-TOF mass spectra of 58 C. glabrata isolates originating from Marseilles (France) and Tunis (Tunisia) as well as collection strains from diverse geographic origins were analyzed. The same analysis was conducted on a subset of C. glabrata isolates that were either susceptible (MIC ≤ 8 mg/l) or resistant (MIC ≥ 64 mg/l) to fluconazole.According to the seminal results, both MALDI-TOF and MLP classifications could highlight C. glabrata population structures associated with either geographical dispersal barriers (p < 10(-5)) or the selection of antifungal drug resistance traits (<10(-5)).In conclusion, MALDI-TOF geographical clustering was congruent with MPL genotyping and highlighted a significant population genetic structure according to fluconazole susceptibility in C. glabrata. Furthermore, although MALDI-TOF and MLP resulted in distinct classifications, MALDI-TOF also classified the isolates with respect to their fluconazole susceptibility profile. Further prospective studies are required to evaluate the capacity of MALDI-TOF typing to investigate C. glabrata infection outbreaks and predict the antifungal susceptibility profile of clinical laboratory isolates.

Development of a novel matrix-assisted laser desorption/ionization time-of-flight mass spectrum (MALDI-TOF-MS)-based typing method to identify meticillin-resistant Staphylococcus aureus clones

Mass spectrum analysis enables species- and subspecies-level identification, and can be used as an epidemiological tool in outbreak management. However, its reliability at clonal level has yet to be established. To establish a matrix-assisted laser desorption/ionization time-of-flight mass-spectrum-based method that enables bacterial clone identification with accuracy equivalent to pulsed-field gel electrophoresis/phage open-reading frame typing (PFGE/POT). Meticillin-resistant Staphylococcus aureus (MRSA) was used in this study. Mass spectra were obtained from a standard strain of S.aureus (ATCC29213) and 57 clinically isolated strains, categorized according to POT. Peaks associated with MRSA clone identification (N=67) were extracted. Based on this peak information, the feasibility of MRSA clone identification was examined by cluster analysis. In addition to the 58 strains used for peak extraction, mass spectrum analysis of 24 clinically isolated outbreak strains revealed that peak data could be used for successful identification of clones. These typing results were fully consistent with the PFGE and POT results. This novel method enables simple and rapid typing with accuracy equivalent to PFGE/POT. This method would be suited to rapid outbreak analysis, offering accurate information to combat infectious diseases.

Comparison of MALDI-TOF mass spectra with microsatellite length polymorphisms in Candida albicans.

Candida albicans is the most frequent yeast involved in human infections. Its population structure can be divided into several genetic clades, some of which have been associated with antifungal susceptibility. Therefore, detecting and monitoring fungal clones in a routine laboratory setting would be a major epidemiological advance. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra results are now widely used as bar codes to identify microorganisms in clinical microbiology laboratories. This study aimed at testing MALDI-TOF mass spectra bar codes to identify clades among a set of C. albicans isolates. Accordingly, 102 clinical strains were genotyped using 10 microsatellite markers and analyzed via MALDI-TOF mass spectrometry. The mass spectra were compared with a reference spectral library including 33 well-characterized collection strains, using a Microflex(TM) system and Biotyper(TM) software, to test the capacity of the spectrum of a given isolate to match with the reference mass spectrum of an isolate from the same genetic clade. Despite high confidence species identification, the spectra failed to significantly match with the corresponding clade (p = 0.74). This was confirmed with the MALDI-TOF spectra similarity dendrogram, in which the strains were dispersed irrespective of their genetic clade. Various attempts to improve intra-clade spectra recognition were unsuccessful. In conclusion, MALDI-TOF mass spectra bar code analysis failed to reliably recognize genetically related C. albicans isolates. Further studies are warranted to develop alternative MALDI-TOF mass spectra analytical approaches to identify and monitor C. albicans clades in the routine clinical laboratory.

Evaluation of structural and functional properties of protein–EGCG complexes and their ability of stabilizing a model β-carotene emulsion

The objectives of the present study were to first covalently modify different bovine milk proteins (α-lactalbumin, β-lactoglobulin, lactoferrin and sodium caseinate) using (−)-epigallocatechin-3-gallate (EGCG), then compare structural and functional properties between covalent and non-covalent protein–EGCG complexes and finally test the complexes with greater antioxidant potential in stabilizing a model β-carotene emulsion. Covalent modification of milk proteins with EGCG was testified by a reduction of free amino groups and free sulfhydryl groups as well as matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS). Furthermore, the proposed schematic formation pathway showed that milk proteins formed covalent complexes with EGCG dimers. Then structural and functional properties of covalent and non-covalent protein–EGCG complexes were analyzed. Covalent protein–EGCG complexes had higher denaturation temperatures than non-covalent ones. Besides, covalent protein–EGCG complexes exhibited much stronger antioxidant activity than the same amount of non-covalent ones. A comparison of interfacial concentration fraction of protein and EGCG in emulsions revealed that covalent protein–EGCG complexes exhibited better interfacial adsorption behavior and greater antioxidant potential than non-covalent ones in emulsion systems. Thus, research was then focused on testing covalent protein–EGCG complexes in a model β-carotene emulsion. Covalent protein–EGCG complexes significantly enhanced chemical stability of β-carotene in emulsions against heat treatment and ultraviolet (UV) light exposure. Given both physical and chemical stability of emulsions, the overall ability of covalent protein–EGCG complexes in stabilizing a model β-carotene emulsion followed the order: sodium caseinate > β-lactoglobulin > lactoferrin > α-lactalbumin.

The application of Gaussian mixture models for signal quantification in MALDI-TOF mass spectrometry of peptides.

Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) coupled with stable isotope standards (SIS) has been used to quantify native peptides. This peptide quantification by MALDI-TOF approach has difficulties quantifying samples containing peptides with ion currents in overlapping spectra. In these overlapping spectra the currents sum together, which modify the peak heights and make normal SIS estimation problematic. An approach using Gaussian mixtures based on known physical constants to model the isotopic cluster of a known compound is proposed here. The characteristics of this approach are examined for single and overlapping compounds. The approach is compared to two commonly used SIS quantification methods for single compound, namely Peak Intensity method and Riemann sum area under the curve (AUC) method. For studying the characteristics of the Gaussian mixture method, Angiotensin II, Angiotensin-2-10, and Angiotenisn-1-9 and their associated SIS peptides were used. The findings suggest, Gaussian mixture method has similar characteristics as the two methods compared for estimating the quantity of isolated isotopic clusters for single compounds. All three methods were tested using MALDI-TOF mass spectra collected for peptides of the renin-angiotensin system. The Gaussian mixture method accurately estimated the native to labeled ratio of several isolated angiotensin peptides (5.2% error in ratio estimation) with similar estimation errors to those calculated using peak intensity and Riemann sum AUC methods (5.9% and 7.7%, respectively). For overlapping angiotensin peptides, (where the other two methods are not applicable) the estimation error of the Gaussian mixture was 6.8%, which is within the acceptable range. In summary, for single compounds the Gaussian mixture method is equivalent or marginally superior compared to the existing methods of peptide quantification and is capable of quantifying overlapping (convolved) peptides within the acceptable margin of error.