Solid-phase microextraction of volatile compounds from flowers of two Brunfelsia species

Two plant species belonging to the genus Brunfelsia, commonly known as “Yesterday-Today-and-Tomorrow”, are closely related, however, differ by their flower fragrance. Flowers of Brunfelsia australis present a pleasant fragrance, whereas flowers of Brunfelsia pauciflora are scentless. SPME/GC/MS analysis on flower samples of both species of Brunfelsia indicated that flowers of B. australis emitted a fresh flowery fragrance, essentially comprising two monoterpenic compounds (linalool and (E)-ocimene). B. pauciflora, on the other hand, produced only a few sesquiterpenoids. These results are considered in an ecological and evolutionary context.     

Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G

The Staphylococcus aureus surface protein G (SasG) is an important mediator of biofilm formation in virulent S. aureus strains. A detailed analysis of its primary sequence has not been reported to date. SasG is highly abundant in the cell wall of the vancomycin-intermediate S. aureus strain HIP5827, and was purified and subjected to sequence analysis by MS. Data from MALDI-TOF and LC-MS/MS experiments confirmed the predicted N-terminal signal peptide cleavage site at residue A₅₁ and the C-terminal cell wall anchor site at residue T₁₀₈₆. The protein was also derivatized with N-succinimidyloxycarbonyl-methyl-tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) to assess the presence of additional N-terminal sites of mature SasG. TMPP-derivatized SasG peptides featured m/z peaks with a 572 Da mass increase over the equivalent underivatized peptides. Multiple N-terminal peptides, all of which were observed in the 150 amino acid segment following the signal peptide cleavage at the residue A₅₁, were characterized from MS and MS/MS data, suggesting a series of successive N-terminal truncations of SasG. A strategy combining TMPP derivatization, multiple enzyme digestions to generate overlapping peptides and detailed MS analysis will be useful to determine and understand functional implications of PTMs in bacterial cell wall-anchored proteins, which are frequently involved in the modulation of virulence-associated bacterial surface properties.     

MALDI-TOF mass spectrometry as a tool for differentiation of Bradyrhizobium species: Application to the identification of Lupinus nodulating strains

Genus Bradyrhizobium includes slow growing bacteria able to nodulate different legumes as well as species isolated from plant tumours. The slow growth presented by the members of this genus and the phylogenetic closeness of most of its species difficults their identification. In the present work we applied for the first time Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to the analysis of Bradyrhizobium species after the extension of MALDI Biotyper 2.0 database with the currently valid species of this genus. With this methodology it was possible to identify strains belonging to phylogenetically closely related species of genus Bradyrhizobium allowing the discrimination among species with rrs gene identities higher than 99%. The application of MALDI-TOF MS to strains isolated from nodules of different Lupinus species in diverse geographical locations allowed their correct identification when comparing with the results of rrs gene and ITS analyses. The nodulation of Lupinus gredensis, an endemic species of the west of Spain, by B. canariense supports the European origin of this species.     

Biochemical and Structural Study of the Homologues of the Thiol-Disulfide Oxidoreductase DsbA in Neisseria meningitidis

Bacterial virulence depends on the correct folding of surface-exposed proteins, a process catalyzed by the thiol-disulfide oxidoreductase DsbA, which facilitates the synthesis of disulfide bonds in Gram-negative bacteria. The Neisseria meningitidis genome possesses three genes encoding active DsbAs: DsbA1, DsbA2 and DsbA3. DsbA1 and DsbA2 have been characterized as lipoproteins involved in natural competence and in host interactive biology, while the function of DsbA3 remains unknown.This work reports the biochemical characterization of the three neisserial enzymes and the crystal structures of DsbA1 and DsbA3. As predicted by sequence homology, both enzymes adopt the classic Escherichia coli DsbA fold. The most striking feature shared by all three proteins is their exceptional oxidizing power. With a redox potential of − 80 mV, the neisserial DsbAs are the most oxidizing thioredoxin-like enzymes known to date. Consistent with these findings, thermal studies indicate that their reduced form is also extremely stable. For each of these enzymes, this study shows that a threonine residue found within the active-site region plays a key role in dictating this extraordinary oxidizing power. This result highlights how residues located outside the CXXC motif may influence the redox potential of members of the thioredoxin family.     

Identification of Outer Membrane Proteins Altered in Response to UVC-Radiation in Vibrio parahaemolyticus and Vibrio alginolyticus

Vibrio parahaemolyticus and V. alginolyticus, marine foodborne pathogens, were treated with UVC-radiation (240 J/m²) to evaluate alterations in their outer membrane protein profiles. Outer membrane protein patterns of UVC-irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Altered proteins were identified by mass spectrometry (MS and MS/MS) and analysis revealed that OmpW, OmpA, Long-chain fatty acid transport protein, Outer membrane receptor protein, Putative uncharacterized protein VP0167, Maltoporin (lamB), Polar flagellin B/D, Agglutination protein Peptidoglycan-associated lipoprotein and MltA-interacting protein MipA were appeared, thereby they can be considered as UVC-stress proteins in some vibrios. In addition, expression of OmpK decreased to non-detectable level. Furthermore, we observed a decrease or an increase in the expression level of other outer membrane proteins.     

Construction of recombinant neisserial Hsp60 proteins and mapping of antigenic domains

Here we report the cloning and expression, in Escherichia coli, of PCR-amplified DNA encoding the 63-kDa stress-inducible protein of Neisseria gonorrhoeae strains VP1 and PiD2, Neisseria meningitidis 2996 and the commensal Neisseria flavescens. DNA sequence analysis revealed in all cases one open reading frame of 541-544 amino acids corresponding to a protein of approximately 57 000 Da. The various neisserial proteins were >98% identical at the amino acid level and showed extensive homology with proteins belonging to the HspSO heat-shock-protein family. We constructed defined glutathione S-transferase fusion polypeptides of the gonococcal Hsp60 homologue to locate antigenic domains on the recombinant protein. Variation in the immunoreactivity of two monoclonal antibodies recognizing a conserved and a neisseria-unique antigenic Hsp60 determinant, respectively, could thus be deduced to result from single amino acid substitutions. Analysis of the antibody response in patients’sera demonstrated reactivity with the same fusion polypeptides in six out of nine sera, indicating that neisserial Hsp60 is expressed during the natural infection and that distinct domains on the protein are immunodominant in vivo.     

Differentiation of species of the Streptococcus bovis/equinus-complex by MALDI-TOF Mass Spectrometry in comparison to sodA sequence analyses

The Streptococcus bovis/equinus complex is a heterogeneous group within the group D streptococci with important clinical relevance regarding infective endocarditis, sepsis and colon carcinoma. The taxonomic identification of species and sub-species of this complex, by the standard methods remains difficult.In the present study, we compared the cluster analysis of 88 strains of species of the S. bovis/equinus complex by sequence analysis of the manganese-dependent superoxide dismutase gene (sodA) and by Matrix Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS). We observed a high congruence of strain grouping by MALDI-TOF MS in comparison with sodA sequence analyses, demonstrating the accuracy and reliability of MALDI-TOF MS in comparison to DNA sequence-based method.By generating mass spectra for each species and sub-species, we were able to discriminate all members of the S. bovis/equinus complex. Furthermore, we demonstrated reliable identifications to the species level by MALDI-TOF MS, independently of cultivation conditions.     

Cluster Analysis of Longidorus Species (Nematoda: Longidoridae), a New Approach in Species Identification

Hierarchical cluster analysis based on female morphometric character means including body length, distance from vulva opening to anterior end, head width, odontostyle length, esophagus length, body width, tail length, and tail width were used to examine the morphometric relationships and create dendrograms for (i) 62 populations belonging to 9 Longidorus species from Arkansas, (ii) 137 published Longidorus species, and (iii) 137 published Longidorus species plus 86 populations of 16 Longidorus species from Arkansas and various other locations by using JMP 4.02 software (SAS Institute, Cary, NC). Cluster analysis dendograms visually illustrated the grouping and morphometric relationships of the species and populations. It provided a computerized statistical approach to assist by helping to identify and distinguish species, by indicating morphometric relationships among species, and by assisting with new species diagnosis. The preliminary species identification can be accomplished by running cluster analysis for unknown species together with the data matrix of known published Longidorus species.     

Identification and biochemical characterization of a GDSL-motif carboxylester hydrolase from Carica papaya latex

An esterase (CpEst) showing high specific activities on tributyrin and short chain vinyl esters was obtained from Carica papaya latex after an extraction step with zwitterionic detergent and sonication, followed by gel filtration chromatography. Although the protein could not be purified to complete homogeneity due to its presence in high molecular mass aggregates, a major protein band with an apparent molecular mass of 41 kDa was obtained by SDS-PAGE. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (679 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 1029 bp encoding a protein of 343 amino acid residues, with a theoretical molecular mass of 38 kDa. From sequence analysis, CpEst was identified as a GDSL-motif carboxylester hydrolase belonging to the SGNH protein family and four potential N-glycosylation sites were identified. The putative catalytic triad was localised (Ser³⁵-Asp³⁰⁷-His³¹⁰) with the nucleophile serine being part of the GDSL-motif. A 3D-model of CpEst was built from known X-ray structures and sequence alignments and the catalytic triad was found to be exposed at the surface of the molecule, thus confirming the results of CpEst inhibition by tetrahydrolipstatin suggesting a direct accessibility of the inhibitor to the active site.     

Evaluation of VITEK matrix‐assisted laser desorption ionization–time of flight mass spectrometry for identification of anaerobes

Rapid and adequate identification of anaerobic bacterial species still presents a challenge for most diagnostic laboratories, hindering the selection of appropriate therapy. In this study, the identification capacity of 16S rRNA sequence analysis, VITEK 2 (BioMérieux, Lyon, France) compact analysis and VITEK MS‐mediated identification for anaerobic bacterial species was compared. Eighty‐five anaerobic bacterial isolates from 11 provinces in China belonging to 14 genera were identified by these three methods. Differences in identification between these three methods were compared. Consistent identification results were obtained for 54 (54/85, 63.5%) isolates by all three methods, the most discordant results being concentrated in Clostridium XI (n = 8) and Bacteroides fragilis (n = 9) clusters. Using the VITEK MS system, 74 (74/90, 82.2%) isolates were identified as single species consistent with 16S rRNA sequence analysis, which was significantly better than the results obtained with VITEK 2 Compact (P < 0.01). Misidentifications by the Vitek 2 Compact and Vitek MS systems were mainly observed in the Clostridium XI (n = 8)and B. fragilis clusters (n = 9). VITEK MS identified anaerobic bacteria even after they had been exposed to oxygen for a week. Identification by the Vitek MS system was more consistent with 16S rRNA sequence analysis than identification by Vitek 2 Compact. Continuous expansion of the VITEK MS database with rare described anaerobic species is warranted to improve both the efficiency and accuracy of VITEK MS identification in routine diagnostic microbiology.     

Identification of Gram-positive anaerobic cocci by MALDI-TOF mass spectrometry

Gram-positive anaerobic cocci (GPAC) are part of the commensal microbiota of humans and are a phylogenetically heterogeneous group of organisms. To evaluate the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of GPAC, a database was constructed, using reference strains of commonly encountered GPAC and clinical isolates of which the sequence of the 16S rRNA gene was determined. Subsequently, the database was validated by identifying 107 clinical isolates of GPAC. Results were compared with the identifications obtained by 16S sequencing or fluorescent in situ hybridization (FISH). Strains belonging to the same species grouped together, in most cases, by MALDI-TOF MS analyses. Strains with sequence similarities less than 98% to their closest relatives, formed clusters distinct from recognized species in the MALDI-TOF MS dendrogram and, therefore could not be identified. These strains probably represent new species. Only three clinical isolates (2 strains of Finegoldia magna and 1 strain of Anaerococcus vaginalis) could not be identified. For all the other GPAC strains (96/107), reliable identifications were obtained. Therefore, we concluded that MALDI-TOF MS is an excellent tool for the identification of phylogenetically heterogeneous groups of micro-organisms such as GPAC.     

Characterization of intact N- and O-linked glycopeptides using higher energy collisional dissociation

Simultaneous elucidation of the glycan structure and the glycosylation site are needed to reveal the biological function of protein glycosylation. In this study, we employed a recent type of fragmentation termed higher energy collisional dissociation (HCD) to examine fragmentation patterns of intact glycopeptides generated from a mixture of standard glycosylated proteins. The normalized collisional energy (NCE) value for HCD was varied from 30 to 60% to evaluate the optimal conditions for the fragmentation of peptide backbones and glycoconjugates. Our results indicated that HCD with lower NCE values preferentially fragmented the sugar chains attached to the peptides to generate a ladder of neutral loss of monosaccharides, thereby enabling the putative glycan structure characterization. In addition, detection of the oxonium ions enabled unambiguous differentiation of glycopeptides from non-glycopeptides. In contrast, HCD with higher NCE values preferentially fragmented the peptide backbone and, thus, provided information needed for confident peptide identification. We evaluated the HCD approach with alternating NCE parameters for confident characterization of intact N- and O-linked glycopeptides in a single liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. In addition, we applied a novel data analysis pipeline, so-called GlycoFinder, to form a basis for automated data analysis. Overall, 38 unique intact glycopeptides corresponding to eight glycosylation sites (six N-linked and two O-linked sites) were confidently identified from a standard protein mixture. This approach provided concurrent characterization of both the peptide and the glycan, thereby enabling comprehensive structural characterization of glycoproteins in a single LC–MS/MS analysis.     

Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory

Whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640–12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates.     

Purification and structural characterization of a novel antibacterial peptide from Bellamya bengalensis: activity against ampicillin and chloramphenicol resistant Staphylococcus epidermidis

Increasing tendency of clinical bacterial strains resistant to conventional antibiotics has being a great challenge to the public's health. Antimicrobial peptides, a new class of antibiotics is known to have the activity against a wide range of bacteria resistant to conventional antibiotics. An antimicrobial peptide of 1676 Da was purified from Bellamya bengalensis, a fresh water snail, using ultrafiltration and reversed phase liquid chromatography. The effect of this peptide on Staphylococcus epidermidis resistant to ampicillin and chloramphenicol was investigated; the MIC and MBC values were 8 μg/ml and 16 μg/ml, respectively. Complete sequence of the peptide was determined by tandem mass spectrometry (MS/MS). Further, peptide net charge, hydrophobicity and molecular modeling were evaluated in silico for better understanding the probable mechanisms of action. The peptide showed the specificity to bacterial membranes. Hence, this reported peptide revealed a promising candidate to contribute in the development of therapeutic agent for Staphylococcal infections.     

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

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

Screening and Molecular Cloning of a Protective Antigen from the Midgut of Haemaphysalis longicornis

Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3'' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an α-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks.     

pepgrep: A Tool for Peptide MS/MS Pattern Matching

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

Serum biomarkers of Burkholderia mallei infection elucidated by proteomic imaging of skin and lung abscesses

Background

The bacterium Burkholderia mallei is the etiological agent of glanders, a highly contagious, often fatal zoonotic infectious disease that is also a biodefense concern. Clinical laboratory assays that analyze blood or other biological fluids are the highest priority because these specimens can be collected with minimal risk to the patient. However, progress in developing sensitive assays for monitoring B. mallei infection is hampered by a shortage of useful biomarkers.

Results

Reasoning that there should be a strong correlation between the proteomes of infected tissues and circulating serum, we employed imaging mass spectrometry (IMS) of thin-sectioned tissues from Chlorocebus aethiops (African green) monkeys infected with B. mallei to localize host and pathogen proteins that were associated with abscesses. Using laser-capture microdissection of specific regions identified by IMS and histology within the tissue sections, a more extensive proteomic analysis was performed by a technique that combined the physical separation capabilities of liquid chromatography (LC) with the sensitive mass analysis capabilities of mass spectrometry (LC-MS/MS). By examining standard formalin-fixed, paraffin-embedded tissue sections, this strategy resulted in the identification of several proteins that were associated with lung and skin abscesses, including the host protein calprotectin and the pathogen protein GroEL. Elevated levels of calprotectin detected by ELISA and antibody responses to GroEL, measured by a microarray of the bacterial proteome, were subsequently detected in the sera of C. aethiops, Macaca mulatta, and Macaca fascicularis primates infected with B. mallei.

Conclusions

Our results demonstrate that a combination of multidimensional MS analysis of traditional histology specimens with high-content protein microarrays can be used to discover lead pairs of host-pathogen biomarkers of infection that are identifiable in biological fluids.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9079-4) contains supplementary material, which is available to authorized users.     

Bacterial species identification from MALDI-TOF mass spectra through data analysis and machine learning

At present, there is much variability between MALDI-TOF MS methodology for the characterization of bacteria through differences in e.g., sample preparation methods, matrix solutions, organic solvents, acquisition methods and data analysis methods. After evaluation of the existing methods, a standard protocol was developed to generate MALDI-TOF mass spectra obtained from a collection of reference strains belonging to the genera Leuconostoc, Fructobacillus and Lactococcus. Bacterial cells were harvested after 24 h of growth at 28 °C on the media MRS or TSA. Mass spectra were generated, using the CHCA matrix combined with a 50:48:2 acetonitrile:water:trifluoroacetic acid matrix solution, and analyzed by the cell smear method and the cell extract method. After a data preprocessing step, the resulting high quality data set was used for PCA, distance calculation and multi-dimensional scaling. Using these analyses, species-specific information in the MALDI-TOF mass spectra could be demonstrated. As a next step, the spectra, as well as the binary character set derived from these spectra, were successfully used for species identification within the genera Leuconostoc, Fructobacillus, and Lactococcus. Using MALDI-TOF MS identification libraries for Leuconostoc and Fructobacillus strains, 84% of the MALDI-TOF mass spectra were correctly identified at the species level. Similarly, the same analysis strategy within the genus Lactococcus resulted in 94% correct identifications, taking species and subspecies levels into consideration. Finally, two machine learning techniques were evaluated as alternative species identification tools. The two techniques, support vector machines and random forests, resulted in accuracies between 94% and 98% for the identification of Leuconostoc and Fructobacillus species, respectively.