MALDI-TOF MS Assay Research Articles

Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y

Mycoplasma hyopneumoniae is an economically-devastating and geographically-widespread pathogen that colonises ciliated epithelium, and destroys mucociliary function. M. hyopneumoniae devotes ~5% of its reduced genome to encode members of the P97 and P102 adhesin families that are critical for colonising epithelial cilia, but mechanisms to impair mucociliary clearance and manipulate host immune response to induce a chronic infectious state have remained elusive. Here we identified two surface exposed M. hyopneumoniae proteases, a putative Xaa-Pro aminopeptidase (MHJ_0659; PepP) and a putative oligoendopeptidase F (MHJ_0522; PepF), using immunofluorescence microscopy and two orthogonal proteomic methodologies. MHJ_0659 and MHJ_0522 were purified as polyhistidine fusion proteins and shown, using a novel MALDI-TOF MS assay, to degrade four pro-inflammatory peptides that regulate lung homeostasis; bradykinin (BK), substance P (SP), neurokinin A (NKA) and neuropeptide Y (NPY). These findings provide insight into the mechanisms used by M. hyopneumoniae to influence ciliary beat frequency, impair mucociliary clearance, and initiate a chronic infectious disease state in swine, features that are a hallmark of disease caused by this pathogen.

A novel short-term high-lactose culture approach combined with a matrix-assisted laser desorption ionization-time of flight mass spectrometry assay for differentiating Escherichia coli and Shigella species using artificial neural networks.

BackgroundEscherichia coli is currently unable to be reliably differentiated from Shigella species by routine matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. In the present study, a reliable and rapid identification method was established for Escherichia coli and Shigella species based on a short-term high-lactose culture using MALDI-TOF MS and artificial neural networks (ANN).Materials and methodsThe Escherichia coli and Shigella species colonies, treated with (Condition 1)/without (Condition 2) a short-term culture with an in-house developed high-lactose fluid medium, were prepared for MALDI-TOF MS assays. The MS spectra were acquired in linear positive mode, with a mass range from 2000 to 12000 Da and were then compared to discover new biomarkers for identification. Finally, MS spectra data sets 1 and 2, extracted from the two conditions, were used for ANN training to investigate the benefit on bacterial classification produced by the new biomarkers.ResultsTwenty-seven characteristic MS peaks from the Escherichia coli and Shigella species were summarized. Seven unreported MS peaks, with m/z 2330.745, m/z 2341.299, m/z 2371.581, m/z 2401.038, m/z 3794.851, m/z 3824.839 and m/z 3852.548, were discovered in only the spectra from the E. coli strains after a short-term high-lactose culture and were identified as belonging to acid shock protein. The prediction accuracies of the ANN models, based on data set 1 and 2, were 97.71±0.16% and 74.39±0.34% (n = 5), with an extremely remarkable difference (p < 0.001), and the areas under the curve of the receiver operating characteristic curve were 0.72 and 0.99, respectively.ConclusionsIn summary, adding a short-term high-lactose culture approach before the analysis enabled a reliable and easy differentiation of Escherichia coli from the Shigella species using MALDI-TOF MS and ANN.

Salmonella spp. prevalence, antimicrobial resistance and risk factor determination in Colombian swine farms

ABSTRACT: To determine Salmonella spp. prevalence/seroprevalence, antimicrobial resistance patterns and risk factor identification associated with its presence in Colombian swine farms. 504 samples (Faeces, swabs and environment samples) were obtained from 21 farms distributed in four geographical regions in Colombia. Salmonella spp. microbiological and molecular detection were determined by two Salmonella spp. MDS3M™ and MALDI-TOF MS assays, respectively. In addition, for serological evaluation 231 serum samples were analyzed employing ELISA Salmonella Pigtype®-Salmonella Ab (QUIAGEN®). Additionally, 41 isolates were tested for antimicrobial susceptibility using broth microdilution technique (Panel B1016-180 Beckman Coulter NC72®) and verified with WHONET 2016 software. Risk factors were assessed from a survey and analyzed for statistical significance by U Mann-Whitney test. An 8.9% prevalence (n=45) and 38.1% (n=88) seroprevalence were determined. All isolates presented 100% antimicrobial susceptibility against amikacin. However, resistance against penicillin, tetracycline, cefuroxime and trimethoprim/sulfamethoxazole was present in more than 50% of evaluated strains. Risk factors associated with Salmonella spp. presence were surface water use, rough-surfaced on floors, presence of hoppers as feeders and worker’s boots. Bacteria were present in animals and environmental samples from evaluated farms. Animal contact and/or exposure with the microorganism were also evident in obtained serological response. Bacteria presence depended on management practices and infrastructure, likewise antibiotic use, supplemented in the diet may have induced an increase in Salmonella spp. antimicrobial resistance.

Boric-Acid-Functionalized Covalent Organic Framework for Specific Enrichment and Direct Detection of cis-Diol-Containing Compounds by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Design and synthesis of a novel matrix that serves as highly selective adsorption material are significant for the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of small molecules in complicated biosamples. In this work, we presented a facile one-pot strategy for the synthesis of boric-acid-functionalized covalent organic frameworks (B-COFs) by using 2,4,6-trihydroxy-1,3,5-benzenetrialdehyde, benzidine, and 4-aminophenyl-boronic acid as ligands. Compared with bare COFs, the B-COFs have similar crystallinity, specific surface, and well-developed pore structure. The surface area and average pore size of B-COFs were 238.0 m2/g and 1.2 nm, respectively. The resulting material was used as an adsorbent for selective enrichment of cis-diol-containing compounds based on an affinity reaction between phenylboronic acid and cis-diol. Using luteolin, riboflavin, and pyrocatechol as model analytes, the enrichment ability of B-COFs as a matrix was examined by MALDI-TOF MS assay, and its high selectivity against target analytes was obtained in the presence of 100 times more anti-nonspecific compounds than that even in the complicated biosample. The limits of detection for luteolin, riboflavin, and pyrocatechol were as low as fg/mL with B-COF enrichment. The B-COFs were further employed and validated for specific enrichment and direct detection of target analytes with complex samples such as human serum, milk, and Capsicum samples. Large surface area, numerous boric-acid active sites, and super stability make B-COFs with high enrichment capacity, high selectivity and sensitivity, satisfying reproducibility, and excellent applicability in MALDI-TOF MS assays.

Prevalence of mutations in the GJB2, SLC26A4, GJB3, and MT-RNR1 genes in 103 children with sensorineural hearing loss in Shaoxing, China.

Mutations in the GJB2, SLC26A4, GJB3, and MT-RNR1 genes are known to be a common cause of hearing loss. However, the frequency of hot-spot mutations and genotype-phenotype correlations in patients with sensorineural hearing loss (SNHL) has been less frequently reported. We conducted a study of 103 children-56 boys and 47 girls, aged 5 months to 9 years (mean: 4.1 yr)-with SNHL who underwent genetic screening for 20 hot-spot mutations of the GJB2, SLC26A4, GJB3, and MT-RNR1 genes. Mutations were detected by multiple-PCR-based MALDI-TOF MS assay. At least one mutated allele was detected in 48 patients (46.6%), and 30 patients (29.1%) carried pathogenic mutations. Among all the detected mutations, the most common were GJB2 c.235delC and SLC26A4 c.919-2A>G, with allele frequencies of 23.8 and 6.8%, respectively. At least one mutant allele of SLC26A4 was detected in the 13 patients who had an enlarged vestibular aqueduct (EVA). Almost half of the children with SNHL carried a common deafness-related mutation, and nearly one-third carried a pathogenic mutation. The mutations in SLC26A4 were prevalent and correlated strongly with EVA.

MALDI-TOF MS identification of ticks of domestic and wild animals in Algeria and molecular detection of associated microorganisms

Recent studies have reported the reliability of MALDI-TOF MS for arthropod identification, including fresh or alcohol-preserved ticks based on leg-derived mass spectra. The aim of this study was to evaluate the performance of MALDI-TOF MS for the identification of alcohol-preserved Algerian ticks collected from different domestic and wild hosts. Secondly, we conducted a molecular survey to detect the presence of bacterial DNA in all ticks that were previously subjected to MALDI-TOF MS. A total of 2635 ixodid and 1401 argasid ticks belonging to 9 distinct species were collected in nine different regions of northeastern Algeria. The legs of 230 specimens were subjected to MALDI-TOF MS assays. Spectral analysis revealed intra-species similarity and inter-species specificity for the MS spectra, which was consistent with the morphological identification. Blind tests against the in-lab database revealed that 93.48% of the tested specimens were correctly identified. The accuracy of the morphological and MALDI-TOF MS identifications was validated by sequencing the 12S ribosomal RNA gene (rRNA) for 33 specimens and all the ticks were correctly identified. The quantitative PCR screening showed that for 219 tested ticks, 15 were positive for Rickettsia spp., 8 for Borrelia spp. and 17 for Anaplasmataceae. The PCR tests were negative for Coxiella burnetii and Bartonella spp. This study supports MALDI-TOF MS being a reliable tool for the identification of arthropods and brings new data that sheds light on tick species diversity and tick-borne diseases in Algeria.

Mechanistic insights into transferable polymyxin resistance among gut bacteria

Polymyxins such as colistin are antibiotics used as a final line of defense in the management of infections with multidrug-resistant Gram-negative bacteria. Although natural resistance to polymyxins is rare, the discovery of a mobilized colistin resistance gene (mcr-1) in gut bacteria has raised significant concern. As an intramembrane enzyme, MCR-1 catalyzes the transfer of phosphoethanolamine (PEA) to the 1 (or 4')-phosphate group of the lipid A moiety of lipopolysaccharide, thereby conferring colistin resistance. However, the structural and biochemical mechanisms used by this integral membrane enzyme remain poorly understood. Here, we report the modeled structure of the full-length MCR-1 membrane protein. Together with molecular docking, our structural and functional dissection of the complex of MCR-1 with its phosphatidylethanolamine (PE) substrate suggested the presence of a 12 residue-containing cavity for substrate entry, which is critical for both enzymatic activity and its resultant phenotypic resistance to colistin. More importantly, two periplasm-facing helices (PH2 and PH2') of the trans-membrane domain were essential for MCR-1 activity. MALDI-TOF MS and thin-layer chromatography assays provide both in vivo and in vitro evidence that MCR-1 catalyzes the transfer of PEA from the PE donor substrate to its recipient substrate lipid A. Also, the chemical modification of lipid A species was detected in clinical species of bacteria carrying mcr-1 Our results provide mechanistic insights into transferable MCR-1 polymyxin resistance, raising the prospect of rational design of small molecules that reverse bacterial polymyxin resistance, as a last-resort clinical option to combat pathogens with carbapenem resistance.

Characterization of Coagulase-Negative Staphylococci and pheno-genotypic beta lactam resistance evaluation in samples from bovine Intramammary infection

ABSTRACT This study aimed to identify Coagulase-Negative Staphylococci (CoNS) species isolated from bovine mastitis, through phenotypic and PCR-RFLP (Restriction Fragment Length Polymorphism-Polimerase Chain Reaction) methods and to compare both techniques to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique. Among them, the PCR-RFLP method, using a partially conserved sequence of the groEL gene, is a promising alternative, because of its reproducibility and reliability. On the other hand, the proteomic technique MALDI-TOF MS provides an accurate and much faster diagnosis and has been increasingly employed in microbiological identification. The pheno-genotypic profiles of beta-lactam resistance were also investigated, this characterization is important, considering that the use of antimicrobials is a key element for mastitis control in dairy farms. The concordance of the phenotypic, PCR-RFLP and MALDI-TOF MS assays to identify CoNS species was 77,5% (31/40). S. chromogenes was the species most frequently isolated. Antibiotic resistance rate was relatively low, registering values of 25.5% to penicillin, 9.6% to oxacillin and 6.2% to cefoxitin. Resistance to imipenem, cephalotin and amoxicillin+clavulanate was not observed. The mecA gene and its variant were detected in 7.6% and 4,1% of the isolates respectively. The blaZ gene was found in 43.2% of the strains resistant to penicillin.

Expression, polymorphism of IRF5 gene and association with serum cytokine levels in pig

Interferon regulatory factor 5 (IRF5) gene is a crucial transcription factor in immune system and has shown its functionally diverse roles in the regulation of antiviral responses. Previous study revealed the IRF5 gene resides in the reported quantitative trait locus (QTLs) for cytokine levels. In this report, the porcine IRF5 gene was selected to investigate its effect on cytokine level in serum. The mRNA expression of IRF5 was also detected by real time quantitative PCR and the results revealed that IRF5 mRNAs were widely expressed in all analysed tissues except muscle. A genomic variant (g.10764 T > C) in exon 7 of the IRF5 gene was identified by direct sequencing, and then the SNP was genotyped by MALDI-TOF MS assay method in three pig populations. The further association analysis indicated that the SNP was significantly associated with the level of interferon-γ (day 20), interleukin 10 (day 35) and ratio of interferon-γ/interleukin 10 (day 35) in serum (p < .05). An interferon known to be key in immune processes. These results suggested that IRF5 could serve as a promising candidate gene for pig disease resistance breeding.

Biofilm production and beta-lactamic resistance in Brazilian Staphylococcus aureus isolates from bovine mastitis.

Staphylococcus spp. play an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant species due to the production of virulence factors such as slime, which is required for biofilm formation. This study aimed to evaluate biofilm production and its possible relation to beta-lactamic resistance in 20 S. aureus isolates from bovine mastitic milk. The isolates were characterized by pheno-genotypic and MALDI TOF-MS assays and tested for genes such as icaA, icaD, bap, agr RNAIII, agr I, agr II, agr III, and agr IV, which are related to slime production and its regulation. Biofilm production in microplates was evaluated considering the intervals determined along the bacterial growth curve. In addition, to determine the most suitable time interval for biofilm analysis, scanning electron microscopy was performed. Furthermore, genes such as mecA and blaZ that are related to beta-lactamic resistance and oxacillin susceptibility were tested. All the studied isolates were biofilm producers and mostly presented icaA and icaD. The Agr type II genes were significantly prevalent. According to the SEM, gradual changes in the bacterial arrangement were observed during biofilm formation along the growth curve phases, and the peak was reached at the stationary phase. In this study, the penicillin resistance was related to the production of beta-lactamase, and the high minimal bactericidal concentration for cefoxitin was possibly associated with biofilm protection. Therefore, further studies are warranted to better understand biofilm formation, possibly contributing to our knowledge about bacterial resistance in vivo.

Identification and typing of free-living Acanthamoeba spp. by MALDI-TOF MS Biotyper

Over the years, the potential pathogenicity of Acanthamoeba for humans and animals has gained increasing attention from the scientific community. More than 24 species belong to this genus, however only some of them are causative agents of keratitis and encephalitis in humans. Due to technical difficulties in diagnosis, these infections are likely to be under-detected. The introduction of 18S rDNA amplification for the identification of Acanthamoeba has dramatically enhanced diagnosis performances, but the attestation of genotyping requires supplementary sequencing-based procedures.In this study, 15 Acanthamoeba strains were collected and grown on nutrient agar media. Each strain was genotyped by end-point PCR assay for the amplification of the 18S rDNA gene and the genotype was assigned by sequencing analysis through neighbor joining phylogenetic tree. In order to optimize standardization of the MALDI-TOF MS assay, we established the collection time point at the cystic phase. Two strains of each genotype were randomly chosen to customize the biotyper database. For all strains, 24 spectral measurements were acquired and submitted to identification and cluster analysis of spectra. The obtained results highlighted the correct identification of Acanthamoeba strains and the overlapping of spectra dendrogram clusters to the 18S genotype assignations.In conclusion, the MALDI-TOF MS Biotyper revealed the capability to identify and genotype the Acanthamoeba strains, providing a new frontier in the diagnostic identification of amaebae and in taxonomic and phylogenetic studies.

Detection of triazole resistance amongCandidaspecies by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS)

MALDI-TOF MS can rapidly identify microorganisms to the species level and may be able to detect antimicrobial resistance. We evaluated the ability of this technology to detect triazole resistance in Candida species.35 C. albicans, 35 C. glabrata, and 37 C. tropicalis strains were exposed to fluconazole, voriconazole, or posaconazole at two different concentrations plus a drug-free control: a midrange concentration (CLSI clinical breakpoint or epidemiologic cut-off value), and a high concentration (fluconazole 64 μg/ml, voriconazole & posaconazole 16 μg/ml). The MALDI-TOF MS spectra at these concentrations were used to create the individual composite correlation index (CCI) matrices for each isolate. When the CCI of the midrange/highest concentration was lower than that of the midrange/null concentration, the strain was classified as resistant. These results were then compared to the classifications for susceptible or resistant obtained by measuring the MICs according to the CLSI M27-A3 antifungal susceptibility testing (AFST) method.The MALDI-TOF MS assay was able to classify triazole susceptibility against all strains. Overall, essential agreement between MALDI-TOF MS and AFST varied between 54% and 97%, and was highest for posaconazole against C. glabrata. The reproducibility of the MALDI-TOF MS assay varied between 54.3 and 82.9% and was best for fluconazole against C. albicans and posaconazole against C. glabrata. Reproducibility was also higher for C. glabrata isolates compared to C. albicans and C. tropicalis.These results demonstrate that MALDI-TOF MS may be used to simultaneously determine the Candida species and classification as susceptible or resistant to triazole antifungals. Further studies are needed to refine the methodology and improve the reproducibility of this assay.

A monoclonal antibody that tracks endospore formation in the microsporidium Nosema bombycis.

Nosema bombycis, the first identified microsporidium, is a destructive pathogen of the silkworm Bombyx mori and causes severe worldwide economic losses in sericulture. Major microsporidian structural proteins, such as the spore wall protein (SWP), are known to be involved in host invasion. In this study, the reactivity of the monoclonal antibody 2B10 was tested against an endospore protein of N. bombycis with a molecular weight size at 50-kDa, using Western blotting. The antigen was purified after immunoprecipitation and was further identified as EOB13320 according to MALDI-TOF MS assay. We found that EOB13320 locates to the surface of the different developmental stages of the parasite, mostly the sporoblast stage and the mature spore after immunoelectron microscopy examination. EOB13320 was also widely distributed in the developing endospore, especially at the sporoblast stage. This endospore protein also accumulated in the cytoplasm of both the merogony and sporoblast stages. These results imply that EOB13320 detected by monoclonal antibody 2B10 is expressed throughout the life cycle of the parasite, notably during the stage when the endospore is formed, and that this protein is important for spore-coat formation and parasite maintenance. Our study could be instrumental in the understanding of spore wall formation and will help to gain greater insight into the biology of this parasite.

Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of molds of the Fusarium genus.

The rates of infection with Fusarium molds are increasing, and a diverse number of Fusarium spp. belonging to different species complexes can cause infection. Conventional species identification in the clinical laboratory is time-consuming and prone to errors. We therefore evaluated whether matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a useful alternative. The 289 Fusarium strains from the Belgian Coordinated Collections of Microorganisms (BCCM)/Institute of Hygiene and Epidemiology Mycology (IHEM) culture collection with validated sequence-based identities and comprising 40 species were used in this study. An identification strategy was developed, applying a standardized MALDI-TOF MS assay and an in-house reference spectrum database. In vitro antifungal testing was performed to assess important differences in susceptibility between clinically relevant species/species complexes. We observed that no incorrect species complex identifications were made by MALDI-TOF MS, and 82.8% of the identifications were correct to the species level. This success rate was increased to 91% by lowering the cutoff for identification. Although the identification of the correct species complex member was not always guaranteed, antifungal susceptibility testing showed that discriminating between Fusarium species complexes can be important for treatment but is not necessarily required between members of a species complex. With this perspective, some Fusarium species complexes with closely related members can be considered as a whole, increasing the success rate of correct identifications to 97%. The application of our user-friendly MALDI-TOF MS identification approach resulted in a dramatic improvement in both time and accuracy compared to identification with the conventional method. A proof of principle of our MALDI-TOF MS approach in the clinical setting using recently isolated Fusarium strains demonstrated its validity.

Rapid, sensitive and specific detection of OXA-48-like-producing Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The emergence of Enterobacteriaceae producing the class D carbapenemase OXA-48 has become a public health concern, particularly in southern Europe and in the Mediterranean area. The existing MALDI-TOF MS assays that detect carbapenemase activity have disregarded the Enterobacteriaceae producing OXA-48-like enzymes. Here, we assessed the ability of an optimized MALDI-TOF MS imipenem hydrolysis assay to detect OXA-48-like-producing Enterobacteriaceae.We tested 372 non duplicate isolates of Enterobacteriaceae from 9 species, among which 92 harbored a blaOXA-48-like gene and 180 were non-susceptible to ertapenem. The vast majority (98.9%) of the OXA-48-like producers were detected and 97.8% of the ertapenem non-susceptible isolates were correctly classified. From the agar plate to the final result, the turnaround time of the test was 90min.This quick, easy and accurate MALDI-TOF MS test can expedite the implementation of hygiene measures to prevent dissemination of OXA-48-like-producing Enterobacteriaceae and limit outbreaks.

Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay

Activated factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS)-based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetic assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting only the final deacylation portion of the transglutaminase reaction. With the MALDI–TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, Staphylococcus aureus fibronectin binding protein A, and thrombin-activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P−1 substrate position is sensitive to charge character, and the P−2 and P−3 substrate positions are sensitive to the broad FXIIIa substrate specificity pockets. The more distant P−8 to P−11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase.

Biomolecular Characterization of the Levansucrase of Erwinia amylovora, a Promising Biocatalyst for the Synthesis of Fructooligosaccharides

Erwinia amylovora is a plant pathogen that affects Rosaceae, such as apple and pear. In E. amylovora the fructans, produced by the action of a levansucrase (EaLsc), play a role in virulence and biofilm formation. Fructans are bioactive compounds, displaying health-promoting properties in their own right. Their use as food and feed supplements is increasing. In this study, we investigated the biomolecular properties of EaLsc using HPAEC-PAD, MALDI-TOF MS, and spectrophotometric assays. The enzyme, which was heterologously expressed in Escherichia coli in high yield, was shown to produce mainly fructooligosaccharides (FOSs) with a degree of polymerization between 3 and 6. The kinetic properties of EaLsc were similar to those of other phylogenetically related Gram-negative bacteria, but the good yield of FOSs, the product spectrum, and the straightforward production of the enzyme suggest that EaLsc is an interesting biocatalyst for future studies aimed at producing tailor-made fructans.

High-Throughput Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry as an Alternative Approach to Monitoring Drug Resistance of Hepatitis B Virus

Long-term antiviral therapy of chronic hepatitis B virus (HBV) infection can lead to the selection of drug-resistant HBV variants and treatment failure. Moreover, these HBV strains are possibly present in treatment-naive patients. Currently available assays for the detection of HBV drug resistance can identify mutants that constitute ≥5% of the viral population. Furthermore, drug-resistant HBV variants can be detected when a viral load is >10(4) copies/ml (1,718 IU/ml). The aim of this study was to compare matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multitemperature single-strand conformation polymorphism (MSSCP) with commercially available assays for the detection of drug-resistant HBV strains. HBV DNA was extracted from 87 serum samples acquired from 45 chronic hepatitis B (CHB) patients. The 37 selected HBV variants were analyzed in 4 separate primer extension reactions on the MALDI-TOF MS. Moreover, MSSCP for identifying drug-resistant HBV YMDD variants was developed and turned out to be more sensitive than INNOLiPA HBV DR and direct sequencing. MALDI-TOF MS had the capability to detect mutant strains within a mixed viral population occurring with an allelic frequency of approximately 1% (with a specific value of ≥10(2) copies/ml, also expressed as ≥17.18 IU/ml). In our study, MSSCP detected 98% of the HBV YMDD variants among strains detected by the MALDI-TOF MS assay. The routine tests revealed results of 40% and 11%, respectively, for INNOLiPA and direct sequencing. The commonly available HBV tests are less sensitive than MALDI-TOF MS in the detection of HBV-resistant variants, including quasispecies.

Tyrosine nitration of voltage dependent anion channels induced by peroxynitrite alters protein structure and function in vitro

Voltage dependent anion channels (VDAC) are key and abundant outer mitochondrial membrane (OMM) proteins. Peroxynitrite (ONOO−) can induce irreversible tyrosine nitration (Ntyr) of some proteins to alter their structure and function. We showed previously that Ntyr of VDAC occurs in cardiac ischemia and reperfusion (IR) injury due to excess superoxide (O2 •−) reacting with nitric oxide to form endogenous ONOO−. We hypothesized that ONOO− directly induces VDAC specific Ntyr and alters VDAC structure and channel activity. To test this, we exposed recombinant VDAC (rVDAC; rat heart VDAC‐1 full‐length cDNA, GenBank: BC072484, Open Biosystems) to exogenous ONOO− in vitro. We evaluated VDAC Ntyr by Western blot with 3 nitrotyrosine (3‐NT) antibody and examined for sites of Ntyr by MALDI‐TOF MS assay. We found that rVDAC can be Ntyr by as low as 10 μM ONOO−, with a higher band intensity as [ONOO−] is elevated and also that Ntyr of rVDAC induced formation of dimers and trimers. The MS assay showed nitration occurred at either 62Y or 67Y. We monitored channel activity of rVDAC by incorporating rVDAC into planar lipid bilayers. At a test potential of −10 mV, we found that 50 μM ONOO− enhanced rVDAC channel conductance as shown by current amplitudes of −25 pA and −30 pA compared to −18 pA before adding ONOO−. Our results demonstrate that rVDAC can be nitrated by ONOO−, which induces both structural and functional changes in rVDAC.

Liposomes alter thermal phase behavior and composition of red blood cell membranes

Unilamellar liposomes composed of natural phospholipids provide a new promising class of protective agents for hypothermic storage, cryopreservation, or freeze-drying of red blood cells (RBCs). In this study, FTIR spectroscopy, MALDI-TOF MS, and colorimetric assays were used to investigate the effects of liposomes composed of a homologous series of linear saturated phosphatidylcholine phospholipids (18:0; 16:0; 14:0; 12:0) on RBC membranes. RBCs were incubated with liposomes at 37°C and both the liposomal and the RBC fraction were analyzed after incubation. FTIR studies showed that liposomes composed of short acyl chain length lipids cause an increase in RBC membrane conformational disorder at suprazero temperatures, whereas long acyl chain length lipids were found to have little effects. The increased lipid conformational disorder in the RBC membranes coincided with a decrease in the cholesterol-to-phospholipid ratio. The opposite effects were found in the liposomes after incubation with RBCs. MALDI-TOF MS analysis showed the presence of short acyl chain length lipids (14:0 and 12:0) in RBC membranes after incubation, which was not observed after incubation with liposomes containing long acyl chain length lipids (18:0 and 16:0). Liposomes alter RBC membrane properties by cholesterol depletion and lipid addition.