Intact Cell Matrix-assisted Laser Research Articles

Novel chip-based isoelectric focusing device for fractionation of bacteria prior to their mass spectrometry identification

We have developed a planar chip utilizing divergent geometry of separation channel capable of vertical free-flow electrophoresis of particles at flows of lower hundreds of microliters per minute. The divergent flow isoelectric focusing (DF-IEF) chip consists of two sheets of clear polystyrene glass which serve as a base with working channels and a top cover sealing the separation channel. Optimization showed that the chip is capable to form pH gradient within 1 h and separation is completed in 5 or more minutes depending on the sample volume. The vertical position of the chip enabled analysis of sedimenting particles including microorganisms. Four different common bacteria species inactivated with H2O2 vapors were analyzed in a series of experiments. Isoelectric points were determined with capillary isoelectric focusing with following fractionation using DF-IEF with intact cell matrix-assisted laser desorption/ionization mass spectrometry detection. The DF-IEF chip fractionation proved promising for bacterial sample preparation from complex matrices for subsequent identification of whole cells by mass spectrometry.

MALDI-TOF MS and genomic analysis can make the difference in the clarification of canine brucellosis outbreaks

Brucellosis is one of the most common bacterial zoonoses worldwide affecting not only livestock and wildlife but also pets. Canine brucellosis is characterized by reproductive failure in dogs. Human Brucella canis infections are rarely reported but probably underestimated due to insufficient diagnostic surveillance. To improve diagnostics, we investigated dogs in a breeding kennel that showed clinical manifestations of brucellosis and revealed positive blood cultures. As an alternative to the time-consuming and hazardous classical identification procedures, a newly developed species-specific intact-cell matrix-assisted laser desorption/ionization–time of flight mass spectrometry analysis was applied, which allowed for rapid identification of B. canis and differentiation from closely related B. suis biovar 1. High-throughput sequencing and comparative genomics using single nucleotide polymorphism analysis clustered our isolates together with canine and human strains from various Central and South American countries in a distinct sub-lineage. Hence, molecular epidemiology clearly defined the outbreak cluster and demonstrated the endemic situation in South America. Our study illustrates that MALDI-TOF MS analysis using a validated in-house reference database facilitates rapid B. canis identification at species level. Additional whole genome sequencing provides more detailed outbreak information and leads to a deeper understanding of the epidemiology of canine brucellosis.

Intrinsic alterations in peripheral neutrophils from cystic fibrosis newborn piglets

BackgroundThe hallmark of the cystic fibrosis (CF) lung disease is a neutrophil dominated lung environment that is associated to chronic lung tissue destruction and ultimately the patient's death. It is unclear whether the exacerbated neutrophil response is primary related to a defective CFTR or rather secondary to chronic bacterial colonization and inflammation. Here, we hypothesized that CF peripheral blood neutrophils present intrinsic alteration at birth before the start of an inflammatory process. MethodsPeripheral blood neutrophils were isolated from newborn CFTR+/+ and CFTR−/− piglets. Neutrophils immunophenotype was evaluated by flow cytometry. Lipidomic and proteomic profile were characterized by liquid chromatography/tandem mass spectrometry (LC-MS/MS), intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) followed by top-down high-resolution mass spectrometry (HRMS), respectively. The ability of CF neutrophils to kill pseudomonas aeruginosa was also evaluated. ResultsPolyunsaturated fatty acid metabolites analysis did not show any difference between CFTR+/+ and CFTR−/− neutrophils. On the other hand, a predictive mathematical model based on the ICM-MS proteomic profile was able to discriminate between both genotypes. Top-down proteomic analysis identified 19 m/z differentially abundant masses that corresponded mainly to proteins related to the antimicrobial response and the generation of reactive oxygen species (ROS). However, no alteration in the ability of CFTR−/− neutrophils to kill pseudomonas aeruginosa in vitro was observed. ConclusionsICM-MS demonstrated that CFTR−/− neutrophils present intrinsic alterations already at birth, before the presence of any infection or inflammation.

Intact cell MALDI-TOF mass spectrometry, a promising proteomic profiling method in farm animal clinical and reproduction research

The objective of this review is to provide new insights into the possible use of a proteomic method known as Intact Cell Matrix-Assisted Laser Desorption-ionization Time-Of-Flight Mass Spectrometry (ICM-MS) in animal clinical research. Here, we give an overview of the basics of this technique, its advantages and disadvantages compared with other proteomic approaches, past applications and future perspectives. A special emphasis on its implementation in animal reproduction science is given, including examples of the reliable use of ICM-MS on fertility screening. In mammals, the ICM-MS profiles from pig epididymal spermatozoa reflect the proteome changes that they undergo during epididymal maturation and could be associated with the acquisition of fertilizing ability. In chicken, using adequate pre-processing and bioinformatics analysis tools, sperm ICM-MS profiles showed characteristic spectral features that allowed their classification according to their actual fertilizing ability. The association of ICM-MS and Top-down proteomic strategies allowed the identification of chicken fertility biomarkers candidates such as protein vitelline membrane outer layer protein 1 (VMO-1) and avian beta-defensin 10 (AvBD10). In female reproduction, a similar approach on ovarian follicular cells allowed the identification of specific markers of oocyte maturation in the oocyte and surrounding cumulus cells. Altogether, these results indicate that ICM-MS profiling could be a suitable approach for molecular phenotyping of male and female gametes.

MALDI-TOF MS portrait of emetic and non-emetic Bacillus cereus group members.

The number of foodborne intoxications caused by emetic Bacillus cereus isolates has increased significantly. As such, rapid and reliable methods to identify emetic strains appear to be clinically relevant. In this study, intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to differentiate emetic and non-emetic bacilli. The phyloproteomic clustering of 34 B. cereus emetic and 88 non-emetic isolates classified as B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus mycoides, showed (i) a clear separation of both groups at a similarity level of 43%, and (ii) a high relatedness among the emetic isolates (similarity of 78%). Specifically, 83 mass peak classes were recognized in the spectral window range between m/z 4000 and 12000 that were tentatively assigned to 41 protein variants based on a bioinformatic approach. Mass variation between the emetic and the non-emetic subsets was recorded for 27 of them, including ten ribosomal subunit proteins, for which inter-strain polymorphism was confirmed by gene sequencing. Additional peaks were assigned to other proteins such as small acid soluble proteins, cold shock proteins and hypothetical proteins, e.g., carbohydrate kinase. Moreover, the results were supported by in silico analysis of the biomarkers in 259 members of B. cereus group, including Bacillus anthracis, based on their whole-genome sequences. In conclusion, the proteomic profiling by MALDI-TOF MS is a promising and rapid method for pre-screening B. cereus to identify medically relevant isolates and for epidemiologic purposes.

Microbial Analysis of Escherichia coli ATCC, Lactobacteria and Saccharomyces cerevisiae Using Capillary Electrophoresis Approach.

Rapid detection and identification of microorganisms is a challenging and important aspect in many areas of our life, beginning with medicine, ending with industry. Unfortunately, classical methods of microorganisms identification are based on time-consuming and labor-intensive approaches. Screening techniques require rapid and cheap grouping of bacterial isolates; however, modern bioanalytics demands comprehensive bacterial studies on molecular level. The new approach to the rapid identification of bacteria is to use the electromigration techniques, especially capillary zone electrophoresis (CZE). CZE is an important technique used in the analysis of microorganisms. However, the analysis of microbial complexes using this technology still encounters several problems-uncontrolled aggregation and/or adhesion to the capillary surface. One way to resolve this issue is the CZE analysis of microbial cell with surface charge modification by bivalent metal ions (e.g., Ca(2+) aq, Zn aq). Under the above conditions, bacterial cells create compact aggregates, and fewer high-intensity signals are observed in electropherograms. The chapter presents the capillary electrophoresis of microbial aggregates approach with UV and one-dimensional intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (ICM MS) detection.

Aluminium foil as a single-use substrate for MALDI-MS fingerprinting of different melanoma cell lines.

Herein, we present the intact cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the fingerprinting of human melanoma cancer cell lines grown on aluminium foil. To perform the MALDI-MS assay, melanoma cells were cultured on a flat and thin foil, which was directly transferred to the target plate of MALDI-MS for analysis. The influence of a wide range of cell fixation protocols (i.e. formalin-based and alcohol-based methods) and MALDI matrices on the obtained characteristic spectra was investigated. For the optimization of the MALDI-MS protocol, the MS fingerprints of the melanoma WM-239 cell line with and without an overexpressed enhanced green fluorescent protein were employed. The fingerprints obtained from WM-239 cells grown on aluminium foil were compared with the intact cell MALDI-MS of the cell pellet and presented higher sensitivity in a high m/z range. The optimized protocol was subsequently applied to characterise melanoma cell lines derived from different cancer stages and allowed identification of unique MS signals that could be used for differentiation between the studied cell lines (i.e. molecular weight equal to 10.0 kDa and 26.1 kDa).

Assignment of functional groups in Gram-positive bacteria

The chemical composition, molecular structure and physicochemical properties of five Gram-positive bacterial strain: Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus, Micococcus luteus were investigated by Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), NMR spectroscopy and intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IC MALDI TOF MS). An analysis of FTIR spectra as a function of pH revealed the three major types of cell wall functional groups – carboxyl group, amino group and phosphate group. An analysis of XPS spectra was determinate the major surface components of bacterial cell. 13C NMR and IC MALDI TOF MS spectra of six bacterial species were registered. Our findings indicate that chemical and structural differences in the cell composition of Gram-positive bacteria can be detected. The obtained results also demonstrate that the combination of FTIR, XPS and NMR spectroscopy with IC MALDI TOF MS technique yields useful information and complements other biochemical and physical methods of microbial cells characteristics.

Evaluation of Intact Cell Matrix-Assisted Laser Desorption/Ionization Timeof- Flight Mass Spectrometry for Capillary Electrophoresis Detection of Controlled Bacterial Clumping

Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a technique of choice for quick classification of bacterial cells and precise characterization of microorganisms. The evaluated MALDI MS procedure was confronted with automated biochemical method using the VITEK 2 Compact system employed as a reference method. Moreover, the investigation aimed at describing and optimization of the environmental conditions having an impact on reproducibility and quality of one-dimensional intact-cell MALDI TOF spectra. The work presents off-line combination of capillary electrophoresis of microbial clumping with spectrometric detection.

Interlaboratory Comparison of Intact-Cell Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Results for Identification and Differentiation of Brucella spp

Classical microbiological diagnosis of human brucellosis is time-consuming, hazardous, and subject to variable interpretation. Intact-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for the routine identification of Brucella spp. Analysis of mass peak patterns allowed accurate identification to the genus level. However, statistical models based on peak intensities were needed for definite species differentiation. Interlaboratory comparison confirmed the reproducibility of the results.

Matrix assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry for identification of plant pathogenic Alternaria species

Identification of microorganisms by intact cell matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technology. In this work, intact MALDI-TOF MS was applied for rapid identification of plant pathogenic Alternaria species based on analysis of protein markers. An analysis of 60 isolates belonging to 12 Alternaria spp. showed characteristic protein finger prints that were specific at species level. When mass fragment data were subjected to partial least squares regression (PLS-R), all these 12 species formed separate groups. The method therefore permits rapid and direct identification of Alternaria spp. from small quantities of fungal mycelium.

Significant Natural Product Biosynthetic Potential of Actinorhizal Symbionts of the Genus Frankia, as Revealed by Comparative Genomic and Proteomic Analyses

Bacteria of the genus Frankia are mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genus Frankia has largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences of Frankia strains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from other Frankia strains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis of Frankia sp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed in Frankia sp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in the Frankia genus.

Rapid identification of Burkholderia cepacia complex species including strains of the novel Taxon K, recovered from cystic fibrosis patients by intact cell MALDI-ToF mass spectrometry

Two approaches based on intact cell matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (IC-MALDI-ToF MS) have been evaluated in order to discriminate and identify nine former Burkholderia cepacia complex (Bcc) species, Burkholderia contaminans belonging to the novel Taxon K, Burkholderia gladioli, and the most relevant non-fermentative (NF) Gram-negative rods recovered from cystic fibrosis (CF) sputum cultures. In total, 146 clinical isolates and 26 reference strains were analysed. IC mass spectra were obtained with high reproducibility applying a recently developed inactivation protocol which is based on the extraction of microbial proteins by trifluoroacetic acid (TFA). In a first approach, spectral analysis was carried out by means of a gel-view representation of mass spectra, which turned out to be useful to recognize specific identifying biomarker proteins (SIBPs). A series of prominent mass peaks, mainly assigned to constitutively expressed proteins, were selected as SIBPs for identifications at the genus and species level. Two distinctive mass peaks present in B. contaminans spectra (7501 and 7900 Da) were proposed as SIBPs for the identification of this novel species. A second approach of spectral analysis based on data reduction, feature selection and subsequent hierarchical cluster analysis was used to obtain an objective discrimination of all species analysed. Both complementary modalities of analyzing complex IC-MALDI-ToF MS data open the path towards a rapid, accurate and objective means of routine clinical microbiology diagnosis of pathogens from sputum samples of CF patients.

Matrix-assisted laser desorption ionisation-time-of of-flight mass spectrometry of intact cells allows rapid identification of Burkholderia cepacia complex

The present study examined the potential of intact-cell matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid identification of Burkholderia cepacia complex (Bcc) bacteria using an Applied Biosystems 4700 Proteomics Analyser. Two software packages were used to analyse mass profiles based on densitometric curves and peak positions. The 75 strains examined, represented the nine established Bcc species and some commonly misidentified species, closely related or biochemically similar to Bcc and relevant in the context of cystic fibrosis microbiology. All Bcc strains clustered together, separated from non-Bcc strains. Within Bcc, most Bcc strains grouped in species specific clusters, except for Burkholderia anthina and Burkholderia pyrrocinia strains which constituted a single cluster. The present study demonstrates that MALDI-TOF MS is a powerful approach for the rapid identification of Bcc bacteria.

Intact cell matrix‐assisted laser desorption/ionization mass spectrometry as a tool to screen drugs in vivo for regulation of protein expression

Here we demonstrate for the first time the application of intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) to study the regulation of protein expression. This technique can be extended to screen the drugs that inhibit protein synthesis in various diseases. We have used Escherichia coli cells expressing a recombinant glutathione-S-transferase (GST) gene under an arabinose-inducible promoter as a model system. Using ICM-MS analysis, we have detected a 28 kDa peak corresponding to the production of recombinant GST under the arabinose-induced condition. Furthermore, recombinant GST protein was purified by a single-step affinity purification using a glutathione Sepharose 4B affinity column from arabinose-induced E. coli cells. The purified GST protein was found to be a 28 kDa protein by MALDI analysis suggesting the arabinose-induced protein is indeed GST. The regulation of protein expression was studied using glucose as an alternative metabolite. The glucose-mediated regulation of the ara-operon was followed using the ICM-MS technique. All the results obtained from ICM-MS data were validated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The present technique can be extended for in vivo screening of drugs and it holds tremendous potential to discover novel drugs against specific protein expressions in different diseases.

Differentiation of mutans streptococci by intact cell matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

It is difficult to distinguish mutans streptococci on the species level, and even more so on the subspecies level. Intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (ICM) was applied to reference strains of five of the species of the mutans group (Streptococcus criceti, Streptococcus downei, Streptococcus mutans, Streptococcus ratti, Streptococcus sobrinus), nonmutans streptococci (Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, and Streptococcus sanguinis), and 177 mutans streptococci isolated from saliva of 10 children. From the analysis of the reference strains, readily distinguishable ICM mass spectra were obtained for the different species. Based on multivariate statistical analysis, a correct and unambiguous assignment was made of the spectra of 159 isolated mutans streptococci to S. mutans and 16 isolates to S. sobrinus. Two isolates were sorted out and were identified by sequencing of their 16S rRNA genes as Streptococcus anginosus. In addition, ICM indicated a misclassification for some reference strains (AHT, V 100 and E 49) and re-classified AHT and E 49 as S. ratti and V 100 as S. sobrinus. This was confirmed by 16S rDNA sequencing. Based on a statistical similarity analysis of the spectra of reference strains and a quantitative assessment of the reproducibility of ICM, the isolates identified as either S. mutans or S. sobrinus were phenotyped on the subspecies level. In the population of the clinical isolates, 14 unambiguously different S. mutans and three different S. sobrinus phenotypes were detected. ICM proved to be a powerful tool for a differentiation of mutans streptococci down to the subspecies level.

Effects of ion mode and matrix additives in the identification of bacteria by intact cell mass spectrometry.

Protocols for the identification of bacterial cells by intact cell matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (ICM-TOFMS) are presented. A mass range of 500 to 10,000 m/z is used. The use of formic acid and the crown ether 1, 4, 7, 10, 13, 16-hexaoxacyclooctadecane (18-crown-6) is described. Crown ether is useful for removing metal ion adducts, which degrade spectral purity, and formic acid promotes positive ions, improves spectral signal, and, hence, increases identification certainty.