Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry as a useful tool for the rapid identification of wild flea vectors preserved in alcohol

Flea identification is a significant issue because some species are considered as important vectors of several human pathogens that have emerged or re‐emerged recently, such as Bartonella henselae (Rhizobiales: Bartonellaceae) and Rickettsia felis (Rickettsiales: Rickettsiaceae). Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has been evaluated in recent years for the identification of multicellular organisms, including arthropods. A preliminary study corroborated the usefulness of this technique for the rapid identification of fleas, creating a preliminary database containing the spectra of five species of flea. However, longterm flea preservation in ethanol did not appear to be an adequate method of storage in the context of specimen identification by MALDI‐TOF MS profiling. The goal of the present work was to assess the performance of MALDI‐TOF MS in the identification of seven flea species [Ctenocephalides felis (Siphonaptera: Pulicidae), Ctenocephalides canis, Pulex irritans (Siphonaptera: Pulicidae), Archaeopsylla erinacei (Siphonaptera: Pulicidae), Leptopsylla taschenbergi (Siphonaptera: Ceratophyllidae), Stenoponia tripectinata (Siphonaptera: Stenoponiidae) and Nosopsyllus fasciatus (Siphonaptera: Ceratophyllidae)] collected in the field and stored in ethanol for different periods of time. The results confirmed that MALDI‐TOF MS can be used for the identification of wild fleas stored in ethanol. Furthermore, this technique was able to discriminate not only different flea genera, but also the two congeneric species C. felis and C. canis.     

Usefulness and accuracy of MALDI‐TOF mass spectrometry as a supplementary tool to identify mosquito vector species and to invest in development of international database

Arthropod‐borne diseases are important causes of morbidity and mortality. The identification of vector species relies mainly on morphological features and/or molecular biology tools. The first method requires specific technical skills and may result in misidentifications, and the second method is time‐consuming and expensive. The aim of the present study is to assess the usefulness and accuracy of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a supplementary tool with which to identify mosquito vector species and to invest in the creation of an international database. A total of 89 specimens belonging to 10 mosquito species were selected for the extraction of proteins from legs and for the establishment of a reference database. A blind test with 123 mosquitoes was performed to validate the MS method. Results showed that: (a) the spectra obtained in the study with a given species differed from the spectra of the same species collected in another country, which highlights the need for an international database; (b) MALDI‐TOF MS is an accurate method for the rapid identification of mosquito species that are referenced in a database; (c) MALDI‐TOF MS allows the separation of groups or complex species, and (d) laboratory specimens undergo a loss of proteins compared with those isolated in the field. In conclusion, MALDI‐TOF MS is a useful supplementary tool for mosquito identification and can help inform vector control.     

Evaluation of sample preparation methods for MALDI-TOF MS identification of highly dangerous bacteria

Aims: To propose a universal workflow of sample preparation method for the identification of highly pathogenic bacteria by MALDI‐TOF MS. Methods and Results: Fifteen bacterial species, including highly virulent Gram‐positive (Bacillus anthracis and Clostridium botulinum) and Gram‐negative bacteria (Brucella melitensis, Burkholderia mallei, Francisella tularensis, Shigella dysenteriae, Vibrio cholerae, Yersinia pestis and Legionella pneumophila), were employed in the comparative study of four sample preparation methods compatible with MALDI‐TOF MS. The yield of bacterial proteins was determined by spectrophotometry, and the quality of the mass spectra, recorded in linear mode in the range of 2000–20 000 Da, was evaluated with respect to the information content (number of signals) and quality (S/N ratio). Conclusions: Based on the values of protein concentration and spectral quality, the method using combination of ethanol treatment followed by extraction with formic acid and acetonitrile was the most efficient sample preparation method for the identification of highly pathogenic bacteria using MALDI‐TOF MS. Significance and Impact of the Study: The method using ethanol/formic acid generally shows the highest extraction efficacy and the spectral quality with no detrimental effect caused by storage. Thus, this can be considered as a universal sample preparation method for the identification of highly virulent micro‐organisms by MALDI‐TOF mass spectrometry.     

Effect of trapping method on species identification of phlebotomine sandflies by MALDI‐TOF MS protein profiling

Sandflies (Diptera: Psychodidae) (Newstead, 1911) are blood‐feeding insects that transmit human pathogens including Leishmania (Trypanosomatida: Trypanosomatidae) parasites, causative agents of the leishmaniases. To elucidate Leishmania transmission cycles, conclusive identification of vector species is essential. Molecular approaches including matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) protein profiling have recently emerged to complement morphological identification. The aim of this study was to evaluate the effect of the trap type used to collect sandflies, specifically Centers for Disease Control (CDC) light or sticky traps, the two most commonly used in sandfly surveys, on subsequent MALDI‐TOF MS protein profiling. Specimens of five species (Phlebotomus ariasi, Phlebotomus papatasi, Phlebotomus perniciosus, Phlebotomus sergenti, Sergentomyia minuta) collected in periurban and agricultural habitats in southeast Spain were subjected to protein profiling. Acquired protein spectra were queried against an in‐house reference database and their quality assessed to evaluate the trap type effect. The results indicate that trap choice can substantially affect the quality of protein spectra in collected sandflies. Whereas specimens retrieved from light traps produced intense and reproducible spectra that allowed reliable species determination, profiles of specimens from sticky traps were compromised and often did not enable correct identification. Sticky traps should therefore not be used in surveys that deploy MALDI‐TOF MS protein profiling for species identification.     

A comparison of MS/MS‐based,stable‐isotope‐labeled,quantitation performance on ESI‐quadrupole TOF and MALDI‐TOF/TOF mass spectrometers

The peptide‐based quantitation accuracy and precision of LC‐ESI (QSTAR Elite) and LC‐MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ‐labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC‐MALDI spectra. The average protein sequence coverages for LC‐ESI and LC‐MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ‐based expression ratios determined by ProteinPilot from the 57 467 ESI‐MS/MS and 26 085 MALDI‐MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7–6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC‐ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC‐MALDI iTRAQ ratios were rejected. Re‐analysis of an archived LC‐MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS‐based peptide quantitation performance of offline LC‐MALDI was comparable with on‐line LC‐ESI, which required threefold less time. However, offline LC‐MALDI allows the re‐analysis of archived HPLC‐separated samples.     

Comparison of molecular species identification for North Sea calanoid copepods (Crustacea) using proteome fingerprints and DNA sequences

Calanoid copepods play an important role in the pelagic ecosystem making them subject to various taxonomic and ecological studies, as well as indicators for detecting changes in the marine habitat. For all these investigations, valid identification, mainly of sibling and cryptic species as well as early life history stages, represents a central issue. In this study, we compare species identification methods for pelagic calanoid copepod species from the North Sea and adjacent regions in a total of 333 specimens. Morphologically identified specimens were analysed on the basis of nucleotide sequences (i.e. partial mitochondrial cytochrome c oxidase subunit I (COI) and complete 18S rDNA) and on proteome fingerprints using the technology of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). On all three molecular approaches, all specimens were classified to species level indicated by low intraspecific and high interspecific variability. Sequence divergences in both markers revealed a second Pseudocalanus species for the southern North Sea identified as Pseudocalanus moultoni by COI sequence comparisons to GenBank. Proteome fingerprints were valid for species clusters irrespective of high intraspecific variability, including significant differences between early developmental stages and adults. There was no effect of sampling region or time; thus, trophic effect, when analysing the whole organisms, was observed in species‐specific protein mass spectra, underlining the power of this tool in the application on metazoan species identification. Because of less sample preparation steps, we recommend proteomic fingerprinting using the MALDI‐TOF MS as an alternative or supplementary approach for rapid, cost‐effective species identification.     

Detection of Biomarkers of Pathogenic Naegleria fowleri Through Mass Spectrometry and Proteomics

Emerging methods based on mass spectrometry (MS) can be used in the rapid identification of microorganisms. Thus far, these practical and rapidly evolving methods have mainly been applied to characterize prokaryotes. We applied matrix‐assisted laser‐desorption‐ionization‐time‐of‐flight mass spectrometry MALDI‐TOF MS in the analysis of whole cells of 18 N. fowleri isolates belonging to three genotypes. Fourteen originated from the cerebrospinal fluid or brain tissue of primary amoebic meningoencephalitis patients and four originated from water samples of hot springs, rivers, lakes or municipal water supplies. Whole Naegleria trophozoites grown in axenic cultures were washed and mixed with MALDI matrix. Mass spectra were acquired with a 4700 TOF‐TOF instrument. MALDI‐TOF MS yielded consistent patterns for all isolates examined. Using a combination of novel data processing methods for visual peak comparison, statistical analysis and proteomics database searching we were able to detect several biomarkers that can differentiate all species and isolates studied, along with common biomarkers for all N. fowleri isolates. Naegleria fowleri could be easily separated from other species within the genus Naegleria. A number of peaks detected were tentatively identified. MALDI‐TOF MS fingerprinting is a rapid, reproducible, high‐throughput alternative method for identifying Naegleria isolates. This method has potential for studying eukaryotic agents.     

Concurrent Proteomic Fingerprinting and Molecular Analysis of Cyathostomins

Rapid, cost‐effective, efficient, and reliable helminth species identification is of considerable importance to understand host–parasite interactions, clinical disease, and drug resistance. Cyathostomins (Nematoda: Strongylidae) are considered to be the most important equine parasites, yet research on this group is hampered by the large number of 50 morphologically differentiated species, their occurrence in mixed infections with often more than 10 species and the difficulties associated with conventional identification methods. Here, MALDI‐TOF MS, previously successfully applied to identify numerous organisms, is evaluated and compared with conventional and molecular genetic approaches. A simple and robust protocol for protein extraction and subsequent DNA isolation allowing molecular confirmation of proteomic findings is developed, showing that MALDI‐TOF MS can discriminate adult stages of the two closely related cyathostomin species Cylicostephanus longibursatus and Cylicostephanus minutus. Intraspecific variability of proteomic profiles within morphospecies demonstrated an identification of morphospecies with an accuracy of close to 100%. In contrast, three genospecies within C. minutus and sex‐specific profiles within both morphospecies could not be reliably discriminated using MALDI‐TOF MS. In conclusion, MALDI‐TOF MS complemented by the molecular protocol is a reliable and efficient approach for cyathostomin species identification.     

Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been used to discriminate moniliaceous fungal species; however, darkly pigmented fungi yield poor fingerprint mass spectra that contain few peaks of low relative abundance. In this study, the effect of dark fungal pigments on the observed MALDI mass spectra was investigated. Peptide and protein samples containing varying concentrations of synthetic melanin or fungal pigments extracted from Aspergillus niger were analyzed by MALDI–TOF and MALDI–qTOF (quadrupole TOF) MS. Signal suppression was observed in samples containing greater than 250 ng/μl pigment. Microscopic examination of the MALDI sample deposit was usually heterogeneous, with regions of high pigment concentration appearing as black. Acquisition of MALDI mass spectra from these darkly pigmented regions of the sample deposit yielded poor or no [M+H]⁺ ion signal. In contrast, nonpigmented regions within the sample deposit and hyphal negative control extracts of A. niger were not inhibited. This study demonstrated that dark fungal pigments inhibited the desorption/ionization process during MALDI–MS; however, these fungi may be successfully analyzed by MALDI–TOF MS when culture methods that suppress pigment expression are used. The addition of tricyclazole to the fungal growth media blocks fungal melanin synthesis and results in less melanized fungi that may be analyzed by MALDI–TOF MS.     

Culture conditions and sample preparation methods affect spectrum quality and reproducibility during profiling of Staphylococcus aureus with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has emerged as a promising tool to rapidly characterize Staphylococcus aureus. Different protocols have been employed, but effects of experimental factors, such as culture condition and sample preparation, on spectrum quality and reproducibility have not been rigorously examined. We applied MALDI‐TOF MS to characterize a model system consisting of five methicillin‐sensitive (MSSA) and five methicillin‐resistant S. aureus isolates (MRSA) under two culture conditions (agar and broth) and using two sample preparation methods [intact cell method and protein extraction method (PEM)]. The effects of these treatments on spectrum quality and reproducibility were quantified. PEM facilitated increases in the number of peaks and mass range width. Broth cultures further improved spectrum quality in terms of increasing the number of peaks. In addition, PEM increased reproducibility in samples prepared using identical culture conditions. MALDI imaging data suggested that the improvement in reproducibility may result from a more homogeneous distribution of sample associated with the broth/PEM treatment. Broth/PEM treatment also yielded the highest rate (96%) of correct classification for MRSA. Taken together, these results suggest that broth/PEM maximizes the performance of MALDI‐TOF MS to characterize S. aureus.

Significance and Impact of the Study

Two culture conditions (agar or broth) and two sample preparation methods (intact cell or protein extraction) were evaluated for their effects on profiling of Staphylococcus aureus using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Results indicated that MALDI‐enabled profiling of S. aureus is most effective when cultures are grown in broth and processed using a protein extraction‐based approach. These findings should enhance future efforts to maximize the performance of this approach to characterize strains of S. aureus.     

Rapid identification and characterization of Vibrio species using whole‐cell MALDI‐TOF mass spectrometry

Aims: Vibrio identification by means of traditional microbiological methods is time consuming because of the many biochemical tests that have to be performed to distinguish closely related species. This work aimed at evaluating the use of MALDI‐TOF mass spectrometry for the rapid identification of Vibrio (V.) spp. as an advantageous application to rapidly discriminate the most important Vibrio spp. and distinguish Vibrio spp. from closely related bacterial species like Photobacterium damselae and Grimontia hollisae and other aquatic bacteria like Aeromonas spp. Methods and Results: Starting from sub‐colony amounts of pure cultures grown on agar plates, a very simple sample preparation procedure was established and combined with a rapid and automated measurement protocol that allowed species identification within minutes. Closely related species like Vibrio alginolyticus and Vibrio parahaemolyticus or Vibrio cholerae and Vibrio mimicus could thus be differentiated by defining signatures of species‐identifying biomarker ions (SIBIs). As a reference method for species designation and for determination of relationships between strains with molecular markers, partial rpoB gene sequencing was applied. Conclusions: The MALDI‐TOF MS‐based method as well as the rpoB sequence‐based approach for Vibrio identification described in this study produced comparable classification results. The construction of phylogenetic trees from MALDI‐TOF MS and rpoB sequences revealed a very good congruence of both methods. Significance and Impact of the Study: Our results suggest that whole‐cell MALDI‐TOF MS‐based proteometric characterization represents a powerful tool for rapid and accurate classification and identification of Vibrio spp. and related species.     

PAS‐cal: A repetitive peptide sequence calibration standard for MALDI mass spectrometry

Mass spectrometers equipped with matrix‐assisted laser desorption/ionization (MALDI‐MS) require frequent multipoint calibration to obtain good mass accuracy over a wide mass range and across large numbers of samples. In this study, we introduce a new synthetic peptide mass calibration standard termed PAS‐cal tailored for MALDI‐MS based bottom‐up proteomics. This standard consists of 30 peptides between 8 and 37 amino acids long and each constructed to contain repetitive sequences of Pro, Ala and Ser as well as one C‐terminal arginine residue. MALDI spectra thus cover a mass range between 750 and 3200 m/z in MS mode and between 100 and 3200 m/z in MS/MS mode. Our results show that multipoint calibration of MS spectra using PAS‐cal peptides compares well to current commercial reagents for protein identification by PMF. Calibration of tandem mass spectra from LC‐MALDI experiments using the longest peptide, PAS‐cal37, resulted in smaller fragment ion mass errors, more matching fragment ions and more protein and peptide identifications compared to commercial standards, making the PAS‐cal standard generically useful for bottom‐up proteomics.     

Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two‐dimensional gel electrophoresis

Introduction – A variety of sample preparation protocols for plant proteomic analysis using two‐dimensional gel electrophoresis (2‐DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. Objective – This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2‐DE. Methodology – Four sample preparation methods were tested: (1) phenol extraction and methanol–ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid–acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS–PAGE (1‐DE) and 2‐DE. Fifteen selected protein spots were trypsinised and analysed by matrix‐assisted laser desorption/ionisation time‐of‐flight tandem mass spectrometry (MALDI‐TOF‐MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Results – Methods number 3 and 4 resulted in large quantities of protein with good 1‐DE separation and were chosen for 2‐DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. Conclusion – The described sample preparation method allows the proteomic analysis of papaya leaves by 2‐DE and mass spectrometry (MALDI‐TOF‐MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species. Copyright © 2009 John Wiley & Sons, Ltd.     

Spectral clustering in peptidomics studies helps to unravel modification profile of biologically active peptides and enhances peptide identification rate

When studying the set of biologically active peptides (the so‐called peptidome) of a cell type, organ, or entire organism, the identification of peptides is mostly attempted by MS. However, identification rates are often dismally unsatisfactory. A great deal of failed or missed identifications may be attributable to the wealth of modifications on peptides, some of which may originate from in vivo post‐translational processes to activate the molecule, whereas others could be introduced during the tissue preparation procedures. Preliminary knowledge of the modification profile of specific peptidome samples would greatly improve identification rates. To this end we developed an approach that performs clustering of mass spectra in a way that allows us to group spectra having similar peak patterns over significant segments. Comparing members of one spectral group enables us to assess the modifications (expressed as mass shifts in Dalton) present in a peptidome sample. The clustering algorithm in this study is called Bonanza, and it was applied to MALDI‐TOF/TOF MS spectra from the mouse. Peptide identification rates went up from 17 to 36% for 278 spectra obtained from the pancreatic islets and from 21 to 43% for 163 pituitary spectra. Spectral clustering with subsequent advanced database search may result in the discovery of new biologically active peptides and modifications thereof, as shown by this report indeed.     

Improving peptide relative quantification in MALDI‐TOF MS for biomarker assessment

Proteomic profiling by MALDI‐TOF MS presents various advantages (speed of analysis, ease of use, relatively low cost, sensitivity, tolerance against detergents and contaminants, and possibility of automation) and is being currently used in many applications (e.g. peptide/protein identification and quantification, biomarker discovery, and imaging MS). Earlier studies by many groups indicated that moderate reproducibility in relative peptide quantification is a major limitation of MALDI‐TOF MS. In the present work, we examined and demonstrate a clear effect, in cases apparently random, of sample dilution in complex samples (urine) on the relative quantification of peptides by MALDI‐TOF MS. Results indicate that in urine relative abundance of peptides cannot be assessed with confidence based on a single MALDI‐TOF MS spectrum. To account for this issue, we developed and propose a novel method of determining the relative abundance of peptides, taking into account that peptides have individual linear quantification ranges in relation to sample dilution. We developed an algorithm that calculates the range of dilutions at which each peptide responds in a linear manner and normalizes the received peptide intensity values accordingly. This concept was successfully applied to a set of urine samples from patients diagnosed with diabetes presenting normoalbuminuria (controls) and macroalbuminuria (cases).     

Experimental evaluation of protein identification by an LC/MALDI/on-target digestion approach

Tryptic digestion of proteins continues to be a workhorse of proteomics. Traditional tryptic digestion requires several hours to generate an adequate protein digest. A number of enhanced accelerated digestion protocols have been developed in recent years. Nonetheless, a need still exists for new digestion strategies that meet the demands of proteomics for high-throughput and rapid detection and identification of proteins. We performed an evaluation of direct tryptic digestion of proteins on a MALDI target plate and the potential for integrating RP HPLC separation of protein with on-target tryptic digestion in order to achieve a rapid and effective identification of proteins in complex biological samples. To this end, we used a Tempo HPLC/MALDI target plate deposition hybrid instrument (ABI). The technique was evaluated using a number of soluble and membrane proteins and an MRC5 cell lysate. We demonstrated that direct deposition of proteins on a MALDI target plate after reverse-phase HPLC separation and subsequent tryptic digestion of the proteins on the target followed by MALDI TOF/TOF analysis provided substantial data (intact protein mass, peptide mass and peptide fragment mass) that allowed a rapid and unambiguous identification of proteins. The rapid protein separation and direct deposition of fractions on a MALDI target plate provided by the RP HPLC combined with off-line interfacing with the MALDI MS is a unique platform for rapid protein identification with improved sequence coverage. This simple and robust approach significantly reduces the sample handling and potential loss in large-scale proteomics experiments. This approach allows combination of peptide mass fingerprinting (PMF), MS/MS peptide fragment fingerprinting (PPF) and whole protein MS for both protein identification and structural analysis of proteins.     

A dual fluorescent/MALDI chip platform for analyzing enzymatic activity and for protein profiling

Being able to rapidly and sensitively detect specific enzymatic products is important when screening biological samples for enzymatic activity. We present a simple method for assaying protease activity in the presence of protease inhibitors (PIs) by measuring tryptic peptide accumulation on copolymer pMALDI target chips using a dual fluorescence/MALDI‐TOF‐MS read‐out. The small platform of the chip accommodates microliter amounts of sample and allows for rapid protein digestion. Fluorescamine labeling of tryptic peptides is used to indicate the proteolytic activity and is shown to be an affordable, simple process, yielding a strong fluorescence signal with a low background. Subsequent MALDI‐TOF‐MS analysis, performed in the same sample well, or in a parallel well without adding fluorescamine, detects the specific tryptic peptides and provides confidence in the assay. The dual read‐out method was applied to screen the inhibition activity of plant PIs, components of plant defense against herbivores and pathogens. Extracts of PIs from Solanum nigrum and trypsin were applied together to a pMALDI chip on which a suitable substrate was adsorbed. The fluorescence and MALDI‐TOF‐MS signal decrease were associated with the inhibitory effect of the PIs on trypsin. The developed platform can be modified to screen novel protease inhibitors, namely, those potentially useful for treating or preventing infection by viruses, including HIV and hepatitis C.     

Exploring membrane and cytoplasm proteomic responses of Alkalimonas amylolytica N10 to different external pHs with combination strategy of de novo peptide sequencing

Identification of differentially proteomic responses to external pHs would pave an access for understanding of survival mechanisms of bacteria living at extreme pH environment. We cultured Alkalimonas amylolytica N10 (N10), a novel alkaliphilic bacterium found in Lake Chahannor, in media with three different pHs and extracted the correspondent membrane and cytoplasm proteins for proteomic analysis through 2‐DE. The differential 2‐DE spots corresponding to the altered pHs were delivered to MALDI TOF/TOF MS for protein identification. Since the genomic data of strain N10 was unavailable, we encountered a problem at low rate of protein identification with 18.1%. We employed, therefore, a combined strategy of de novo sequencing to analyze MS/MS signals generated from MALDI TOF/TOF MS. A significantly improved rate of protein identification was thus achieved at over than 70.0%. Furthermore, we extensively investigated the expression of these pH‐dependent N10 genes using Western blot and real‐time PCR. The conclusions drawn from immunoblot and mRNA measurements were mostly in agreement with the proteomic observations. We conducted the bioinformatic analysis to all the pH‐dependent N10 proteins and found that some membrane proteins participated in iron transport were differentially expressed as external pH elevated and most of differential proteins with increased or bell‐shape mode of pH‐dependence were involved in bioenergetic process and metabolism of carbohydrates, fatty acid, amino acids, and nucleotides. Our data thus provide a functional profile of the pH‐responsive proteins in alkaliphiles, leading to elucidation of alkaliphilic‐adaptive mechanism.     

Evidence for the presence of Ctenocephalides orientis in livestock dwellings in northwest Iran

Fleas are important vectors of diseases such as murine typhus, tularaemia, hymenolepiasis and plague. The presence of active foci and history of human‐ and flea‐transmitted plague in northwest Iran prompted the present group to collect and identify fleas from human and livestock dwellings across West Azerbaijan Province. Adult fleas were collected and identified using routine taxonomic keys. Species designation was confirmed by sequencing the cytochrome oxidase subunit I (COI). Of the collected specimens (n = 989), 104 were collected off‐host (30 from human dwellings and 74 in light traps) and the rest were found on hosts (107 on animals and 778 by human bait). Of these fleas, 394 (40%) were male and 595 (60%) were female. The collected specimens belonged to the species Ctenocephalides canis, Ctenocephalides felis, Ctenocephalides orientis and Pulex irritans (all: Siphonaptera: Pulicidae). The amplified COI fragment, in addition to confirming the morphological identification of species, showed good efficacy in separating the different species in the phylogenetic analysis. In addition to the identification of fleas from human and livestock dwellings using morphological and molecular characteristics, the current paper represents the first report of the presence of C. orientis in northwest Iran. This finding suggests that changing climate conditions may have expanded the distribution of this species.     

Cofeeding intra‐ and interspecific transmission of an emerging insect‐borne rickettsial pathogen

Cat fleas (Ctenocephalides felis) are known as the primary vector and reservoir of Rickettsia felis, the causative agent of flea‐borne spotted fever; however, field surveys regularly report molecular detection of this infectious agent from other blood‐feeding arthropods. The presence of R. felis in additional arthropods may be the result of chance consumption of an infectious bloodmeal, but isolation of viable rickettsiae circulating in the blood of suspected vertebrate reservoirs has not been demonstrated. Successful transmission of pathogens between actively blood‐feeding arthropods in the absence of a disseminated vertebrate infection has been verified, referred to as cofeeding transmission. Therefore, the principal route from systemically infected vertebrates to uninfected arthropods may not be applicable to the R. felis transmission cycle. Here, we show both intra‐ and interspecific transmission of R. felis between cofeeding arthropods on a vertebrate host. Analyses revealed that infected cat fleas transmitted R. felis to naïve cat fleas and rat fleas (Xenopsylla cheopis) via fleabite on a nonrickettsemic vertebrate host. Also, cat fleas infected by cofeeding were infectious to newly emerged uninfected cat fleas in an artificial system. Furthermore, we utilized a stochastic model to demonstrate that cofeeding is sufficient to explain the enzootic spread of R. felis amongst populations of the biological vector. Our results implicate cat fleas in the spread of R. felis amongst different vectors, and the demonstration of cofeeding transmission of R. felis through a vertebrate host represents a novel transmission paradigm for insect‐borne Rickettsia and furthers our understanding of this emerging rickettsiosis.