Protein Mass Fingerprinting Research Articles

Differentiation and Identification of Endophytic Bacteria from Populus Based on Mass Fingerprints and Gene Sequences.

Plant endophytic bacteria play important roles in plants' growth and resistance to stress. It is important to characterize endophytic bacteria to be able to understand their benefits. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful technique for bacterial identification due to its high throughput and simple procedures. In this study, the endophytic bacteria separated from Populus (the leaves, roots and stems of Populus tomentosa Carrière; stems of Populus nigra Linn. var. nigra; and stems of Populus canadensis Moench) were identified and classified based on MALDI-TOF MS data and 16S rRNA gene sequencing. The sampling and preparation of bacteria were optimized to obtain meaningful protein mass fingerprints. The composite correlation index (CCI) values of the inter-genera and inter-species protein mass fingerprints demonstrated sufficient differences between the strains. In the CCI value matrix for ten species in the same genus, all the CCI values were less than 0.5. Among the species, 95.6% of all the CCI values were less than 0.5. After data processing, the classification capacity of the protein mass fingerprints was verified using inter-specific and inter-generic PCoA. To compare different methods' potential for differentiation and phylogenetic analysis, a dendrogram of the MS profiles and a phylogenetic tree based on the 16S rRNA gene sequences were constructed using 61 endophytic bacteria found in Populus. The clustering and grouping results show that the phylogenetic analysis based on MALDI-TOF MS is similar to that based on 16S rRNA gene sequencing. This study provides a valuable reference for differentiating and identifying endophytic bacteria according to their protein mass fingerprints.

Anti-inflammatory, remorin-like protein from green marine Macroalga Caulerpa sertularioides (S.G.Gmel.) M.Howe

The most prevalent natural source of hydrocolloids, cosmetics, medications, and nutraceuticals is marine seaweed (macroalgae). Numerous bioactivities, including antiviral, anticancer, anti-inflammatory, and immunomodulatory characteristics, have been found in bioactive substances such as polyphenols and sulfated and non-sulfated polysaccharides. As a result, new start-up projects and industries based on seaweed are emerging in all regions of the world with abundant marine biodiversity. In this current investigation, the anti-inflammatory activity of two different marine macroalgae Caulerpa racemosa (CR) and Caulerpa sertularioides (CS) was evaluated. Consequently, CS demonstrated more anti-inflammatory and antioxidant effects at a lower dose than CR. The IC50 value for DPPH inhibition was 456.1 μg/mL, and 180.9 μg/mL for CS and CR respectively. A similar result was obtained in the case of protein denaturation (PD), membrane stabilization (MS), and protease inhibition (PI) anti-inflammatory assays with 127.2 μg/mL, 135.5 μg/mL, and 71.88 μg/mL for CR, and 66.78 μg/mL, 88.96 μg/mL, and 59.54 μg/mL for CS respectively. Based on the SDS-PAGE, the molecular weight of lectin responsible for the anti-inflammatory activity was determined as 17 kDa. Protein mass fingerprinting was performed for the particular lectin by in-gel trypsin digestion, MALDI-MS analysis, and Mascot peptide mass fingerprinting. Because of this, the unidentified lectin protein was discovered to be a remorin-like protein that shared 65% of its sequence with the remorin-like protein of Aegilops tauschii subsp. tauschii. Therefore, it is the hitherto report on the presence of remorin-like protein from the green macroalga Caulerpa sertularioides.

Protein Mass Fingerprinting and Antioxidant Power of Hemp Seeds in Relation to Plant Cultivar and Environment

Cannabis sativa (hemp) seeds are considered a functional food for their favorable contents of essential fatty acids, proteins and antioxidants. Beyond phenolics and carotenoids, the bioactivity of proteins has recently been investigated. However, plant genotype and environmental conditions can affect quantity and quality of macronutrients and phytochemicals in seeds, influencing their nutraceutical properties. In this study, the effects of plant variety and seed origin on the protein profile and antioxidant activity of hemp seeds were evaluated. Seeds from two cultivars, Secuieni Jubileu and Finola, were harvested from a mountain field located in Italy and compared with reference seeds used for sowing. Albumin and globulin extracts were obtained using the Osborne method and their antioxidant power was assayed (DPPH and ABTS methods). A matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry method was developed for protein fingerprinting analysis. Albumins from seeds of the mountain site showed higher radical scavenging activity and compounds of lower molecular weight than reference seeds, suggesting a role of proteins in the observed bioactivity. The MALDI-TOF method discriminated samples according to origin and variety, highlighting changes in the protein profile and identifying signals which could be used as markers of hemp cultivars.

Ribosomal Protein Database Profiling Lends Clarity to Ribosomal Protein Evolution and Mass Distribution

Existence of theoretical ribosomal protein mass fingerprint as well as utility of ribosomal protein as biomarkers in mass spectrometry microbial identification suggests phylogenetic significance for this class of proteins. To serve the above two functions, facile means of identifying and extracting important attributes of ribosomal proteins from proteome data file of microbial species must be found. Additionally, there is a need to calculate important properties of ribosomal proteins such as molecular weight and nucleotide sequence based on amino acid sequence information from FASTA proteome file. This work sought to support the above endeavour through developing MATLAB software that extracts the amino acid sequence information of all ribosomal proteins from the FASTA proteome data file of a microbial species downloaded from UniProt. Built-in functions in MATLAB are subsequently employed to calculate important properties of extracted ribosomal proteins such as number of amino acid residue, molecular weight and nucleotide sequence. All information above are output, as a database, to an Excel file for ease of storage and retrieval. Data available from the analysis of an Escherichia coli K-12 proteome revealed that the bacterium possesses a total of 59 ribosomal proteins distributed between the large and small ribosome subunits. The ribosomal protein ranges in sequence length from 38 (50S ribosomal protein L36) to 557 (30S ribosomal protein S1). In terms of molecular weight distribution, the profiled ribosomal proteins range in weight from 4364.305 Da (50S ribosomal protein L36) to 61157.66 Da (30S ribosomal protein S1). More important, analysis of the distribution of the molecular weight of different ribosomal proteins in E. coli reveals a smooth curve that suggests strong co-evolution of ribosomal protein sequence and mass given the tight constraints that a functional ribosome presents. Finally, cluster analysis reveals a preponderance of small ribosomal proteins compared to larger ones, which remains to be a mystery to evolutionary biologists. Overall, the information encapsulated in the ribosomal protein database should find use in gaining a better appreciation for the molecular weight distribution of ribosomal proteins in a species, as well as delivering information for using ribosomal protein biomarkers in identifying particular microbial species in mass spectrometry microbial identification.

Theoretical Ribosomal Protein Mass Fingerprint Database

Ribosomes are highly conserved macromolecular machines whose critical function is protein synthesis. However, existence of unique molecular mass of the same type of ribosomal protein for individual species in the same domain of life raises the interesting question concerning the interaction between natural selection forces and the conservation of structure and function of ribosomal proteins. Thus, given differentiated molecular mass and sequence of ribosomal proteins across species, the structures of ribosomes are correspondingly differentiated even though the general structure and function of the macromolecular machine is conserved across species in the same domain of life. The collection of molecular mass of all ribosomal proteins in the large and small ribosome subunits can be understood as the ribosomal protein mass fingerprint of the species useful for gaining fundamental knowledge of ribosomal proteins, as well as serving as tools for species identification through comparison of ribosomal protein mass spectra. This preprint introduces the Theoretical Ribosomal Protein Mass Fingerprint database that comprises the theoretical molecular mass of all ribosomal proteins of a species calculated based on available amino acid sequence information of the ribosomal proteins. Using amino acid sequence information from the Ribosomal Protein Gene Database, the Theoretical Ribosomal Protein Mass Fingerprint database (https://ngwenfa.wordpress.com/database/) spans species from cyanobacteria, fungus, bacteria, archaea, nematodes, diatoms, micro-algae, and various model organisms. The database should be useful as a resource for gaining fundamental understanding of the mass distribution of ribosomal proteins of a species, or serving as a limited reference database for identifying species based on comparing experimental ribosomal protein mass fingerprint of unknown species against theoretically calculated ones of known species. Future expansion of the database will aim to catalogue the theoretical ribosomal protein mass fingerprint of more microbial species using amino acid sequence information from UniProt

Influence of High Hydrostatic Pressure on the Identification of Lactobacillus by MALDI-TOF MS- Preliminary Study.

In the present study, we assessed the ability of MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) to identify microbial strains subjected to high hydrostatic pressure (HHP) as a stress factor. Protein changes induced by HHP can affect the identification of microorganisms when the identification technique is based on the protein profile. We evaluated two methods, namely MALDI-TOF MS and 16S rDNA sequencing, as a valuable tool to identify Lactobacillus species isolated from spoiled food, juices and beers. The data obtained from the protein mass fingerprint analysis of some of the lactobacilli strains showed differences in unpressured and pressured mass spectrum profiles (MSPs), which influenced the results of the identification. Four out of 13 strains (30%) showed different MSP results for unpressured and pressured samples and these results did not overlap with the 16S rDNA identification results. The 16S rDNA sequencing method revealed that five unpressured strains (38%) and four pressured strains (40%) were identified correctly by MALDI-TOF MS. Both methods showed compatible results in 38% of unpressured strains and in 30% of pressured strains. Stress factors, cultivation methods or the natural environment from which the bacteria were derived can affect their protein profile and thus change the mass spectrum. It is necessary to expand the database with a wide range of mass spectra dedicated to a high-throughput study of the microorganisms derived from different environments.

Proteomic sift through serum and endometrium profiles unraveled signature proteins associated with subdued fertility and dampened endometrial receptivity in women with polycystic ovary syndrome.

The objective of this study is to discern the proteomic differences responsible for hampering the receptivity of endometrium and subduing the fertility of females with polycystic ovary syndrome in analogy to healthy fertile females. This study was designed in collaboration with Hakeem Abdul Hameed Centenary Hospital affiliated to Jamia Hamdard, New Delhi, India. Serum samples were taken from infertile PCOS subjects (n = 6) and fertile control subjects (n = 6) whereas endometrial tissue samples were recruited from ovulatory PCOS (n = 4), anovulatory PCOS (n = 4) and normal healthy fertile control subjects (n = 4) for proteomic studies. Additionally, endometrial biopsies from healthy fertile control (n = 8), PCOS with infertility (n = 6), unexplained infertility (n = 3) and endometrial hyperplasia (n = 3) were taken for validation studies. Anthropometric, biochemical and hormonal evaluation was done for all the subjects enrolled in this study. Protein profiles were generated through 2D-PAGE and differential proteins analyzed with PD-QUEST software followed by identification with MALDI-TOF MS protein mass fingerprinting. Validation of identified proteins was done through RT-PCR relative expression analysis. Protein profiling of serum revealed differential expression of proteins involved in transcriptional regulation, embryogenesis, DNA repair, decidual cell ploidy, immunomodulation, intracellular trafficking and degradation processes. Proteins involved in cell cycle regulation, cellular transport and signaling, DNA repair, apoptotic processes and mitochondrial metabolism were found to be differentially expressed in endometrium. The findings of this study revealed proteins that hold strong candidature as potential drug targets to regulate the cellular processes implicating infertility and reduced receptivity of endometrium in women with polycystic ovary syndrome.

Tetraconazole alters the methionine and ergosterol biosynthesis pathways in Saccharomyces yeasts promoting changes on volatile derived compounds.

The influence of antifungal tetraconazole residues (either as an active substance or as a commercial formulation product) on the fermentative activity of Saccharomyces cerevisiae yeast was evaluated in pasteurized Garnacha red must by using laboratory-scale fermentation assays. The presence of this fungicide promoted a slight decrease in glucose consumption. Volatile fermentative-derived compounds were evaluated in deep. Statistically significant changes were found in methionol (with a mean decrease of around 24%), fatty acids (with increments ranged from 23% to 66%), and ethyl esters (with increases ranged from 23% to 145%) contents when grape musts were enriched with the commercial formulation at both contamination levels assayed. Based on protein mass fingerprinting analysis, it was possible to relate these variations on volatiles content with changes in the activity of several enzymes (Met3p, Met14p, Adh2p, Hmg1p, Erg5p, Erg6p, Erg11p, and Erg20p) involved in the secondary metabolism of yeasts.

Biperiden is an inhibitor of acetylcholinesterase

Rhizostoma pulmo (Barrel Jellyfish) is one of the commonly found jellyfishes on the South-Goan coast of India. Here we present characterization of R. pulmo tentacle extract. The tentacle extracts were found to be capable of affecting the hemostatic system at three different levels, as it exhibited fibrinogenolysis, fibrinolysis and inhibition of ADP induced platelet aggregation. It preferentially cleaved the Aα chain of fibrinogen, followed by the Bβ chain and the γ chain. The tentacle extract also showed significant hemolytic activity against human RBCs and strong proteolytic activity for substrates like (azo) casein and gelatin. However, this proteolytic activity was completely inhibited by EDTA (metalloproteinase inhibitor) but not by PMSF (serine proteinase inhibitor). The extract was devoid of phospholipase activity. A semi-purified protein possessing fibrinogenolytic activity was obtained by a combination of ammonium sulphate precipitation and size exclusion HPLC. Atomic absorption analysis of this protein indicated presence of Zn2+ and treatment with metalloproteinase inhibitor caused complete loss of activity. A 95 kDa metalloproteinase was identified in this fraction and was named Rhizoprotease. Protein Mass Fingerprinting of Rhizoprotease indicates it to be a novel protein.

Partial purification and identification of a metalloproteinase with anticoagulant activity from Rhizostoma pulmo (Barrel Jellyfish)

Rhizostoma pulmo (Barrel Jellyfish) is one of the commonly found jellyfishes on the South-Goan coast of India. Here we present characterization of R. pulmo tentacle extract. The tentacle extracts were found to be capable of affecting the hemostatic system at three different levels, as it exhibited fibrinogenolysis, fibrinolysis and inhibition of ADP induced platelet aggregation. It preferentially cleaved the Aα chain of fibrinogen, followed by the Bβ chain and the γ chain. The tentacle extract also showed significant hemolytic activity against human RBCs and strong proteolytic activity for substrates like (azo) casein and gelatin. However, this proteolytic activity was completely inhibited by EDTA (metalloproteinase inhibitor) but not by PMSF (serine proteinase inhibitor). The extract was devoid of phospholipase activity. A semi-purified protein possessing fibrinogenolytic activity was obtained by a combination of ammonium sulphate precipitation and size exclusion HPLC. Atomic absorption analysis of this protein indicated presence of Zn2+ and treatment with metalloproteinase inhibitor caused complete loss of activity. A 95 kDa metalloproteinase was identified in this fraction and was named Rhizoprotease. Protein Mass Fingerprinting of Rhizoprotease indicates it to be a novel protein.

Fast and accurate identification of molds and yeast-like fungi directly from positive blood cultures by MALDI-TOF mass spectrometry

Molds and yeast-like fungi are potential life-threatening agents of fungemia in immunocompromised patients. Fast and accurate identification (ID) of these pathogens helps to hasten the best-targeted antifungal therapy, improving the patients’ prognosis. We describe a new strategy that enabled the identification of molds or yeast-like fungi directly from positive blood culture (BC) by MALDI-TOF mass spectrometry (MS). BCs with Gram staining revealing arthroconidia and/or hyphae suggestive of fungemia by genera other than Candida were prospectively selected from January to December 2015. Blood aliquots were submitted to an in-house protein extraction protocol. Briefly, 4 ml aliquot was submitted to a lysis centrifugation step (step a) followed by protein extraction with ethanol and 70 % formic acid (step b). One microliter of the supernatant was spotted in quadruplicate in MALDI-TOF MS target and overlaid with 1 μl of α-cyano-4-hydroxycinnamic acid matrix. The pathogens that grew on Sabouraud dextrose agar (SDA) after 72 hours incubation at 30 °C (standard procedure) were also analyzed by MALDI-TOF MS. A loop full of conidia was suspended in 300 μl of deionized water and processed as in step b above. Protein mass fingerprints were obtained using the MALDI-TOF Vitek MS™ system (bioMérieux) and processed by the research use only (RUO) mode using an extended database [SARAMIS™ (v.4.12) plus in-house database]. ID was valid when a spectrum matched with a SuperSpectrum with a confidence level ≥ 75 %, as defined by the manufacturer. The final IDs were confirmed by PCR and sequence analyses of either the ITS/IGS regions of the ribosomal DNA, or the elongation factor-1 alpha gene. Until December 2015, seven potential non-Candida fungi were identified in blood cultures from six immunocompromised patients and an additional patient under intensive care treatment due to thoracic aortic dissection. In all cases, MALDI-TOF MS gave correct genus ID directly from positive BCs (confidence levels 80–99.9 %). Species ID was achieved in the cases of fungemia by Fusarium solani (n = 2), Trichosporon asahii (n = 2) and Geotrichum clavatum (Saprochaete clavata), being the identification of Exophiala dermatitidis and Fusarium verticillioides restricted to genus level. Identical ID results were obtained from the isolates grown on SDA, but with higher confidence levels (96–99.9 %). Five patients (#1–4 and #7) had targeted antifungal therapy guided by the MALDI-TOF MS species ID directly from positive BCs ; four of them survived (80 %). Of these patients, four had risk factors of poor prognosis such as severe neutropenia (neutrophils < 100 cells/mm3) and/or renal failure ; the remaining (#4) was under chemotherapy and systemic corticosteroids at the moment of the catheter related bloodstream infection. Patient #2 had persistent neutropenia and several positive BCs for Saprochaete clavata, and despite targeted dual antifungal therapy with amphotericin B deoxycholate plus voriconazole promptly introduced after fungal ID, deceased 18 days after the first positive BC. Patients #5 and #6 had Fusarium solani fungemia but MALDI-TOF guided targeted antifungal therapy could not be prescribed in one (#5) because he died before final ID of the fungemia and in the other (#6) because the patient was under palliative treatment ; this patient died subsequently due to a polymicrobial bloodstream infection.

Hsp70 and Theta Subunit of T Complex Protein , a Response to Salt Stress in the Halophyte Sesuvium Portulacastrum

The proteomic analysis was performed to explore the salt responsive proteins in S. portulacastrum a salt marsh euhalophyte. Protein profile showed profound differences in the expression pattern of salt treated plant. The total protein content was significantly high in leaf than in shoot and root. 2D electrophoresis revealed that 7 proteins were down regulated, 6 proteins were up-regulated and 46 proteins were newly expressed in S.portulacastrum leaves. Two spots from newly expressed proteins were randomly selected and characterized by MALDI-TOF/MS analysis. Protein mass fingerprinting annotation showed different peptide intensities and subsequent sequence homology search using MASCOT software categorized those proteins as Hsp70 and theta subunit of T complex protein with pI/kDa, 5.2/70kDa and 4.9/53kDa respectively. Hsp70 has been proposed as biomarker for stress produced by NaCl which emphasize its role in protecting plants against stress, whereas theta subunit of T complex protein seems to have role during protein folding expressed during abiotic stress, which yet to be characterized. The resulting findings addressed the significance of stress related proteins on the survival of plants in extreme conditions and it can be complemented with large scale studies on functional and molecular interaction of proteins expressed during saline stress.

Identification and characterization of a Leishmania donovani serine protease inhibitor: Possible role in regulation of host serine proteases.

This study aims to identify, purify, and characterize an endogenous serine protease inhibitor from an Indian strain of Leishmania donovani, which causes the fatal visceral leishmaniasis. (i) Reverse zymography was used to identify the serine protease inhibitor by inhibiting the gelatinolytic activity of serine protease. (ii) Purification was performed by combining heat treatment, ultracentrifugation, and affinity and gel permeation chromatography. (iii) Spectrophotometric assays were conducted to quantify and compare the inhibitory activity of the L. donovani serine protease inhibitor (LdISP). (iv) Further, the protein was identified by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS). An endogenous inhibitor with an apparent molecular weight of 21.8 kDa, which is acidic in nature, having a pI of 5.9 was identified. The Ki value of the inhibitor for trypsin was determined to be in the nanomolar range. The protein has the following features: (i) ecotin-like nature, (ii) cross-organism functionality, that is, an inhibitory effect on the serine proteases of higher organisms other than its own, and (iii) homology with other such proteins from a different species of Leishmania on conducting protein mass fingerprinting after MALDI ToF MS. The inhibitor shows varying and entirely contrasting efficacies toward serine proteases of its own as well as of higher organisms. This indicates that it accelerates disease progression and drives parasite survival as it inhibits the activities of the host serine proteases.

Metabolic pathway involved in 2-methyl-6-ethylaniline degradation by Sphingobium sp. strain MEA3-1 and cloning of the novel flavin-dependent monooxygenase system meaBA.

2-Methyl-6-ethylaniline (MEA) is the main microbial degradation intermediate of the chloroacetanilide herbicides acetochlor and metolachlor. Sphingobium sp. strain MEA3-1 can utilize MEA and various alkyl-substituted aniline and phenol compounds as sole carbon and energy sources for growth. We isolated the mutant strain MEA3-1Mut, which converts MEA only to 2-methyl-6-ethyl-hydroquinone (MEHQ) and 2-methyl-6-ethyl-benzoquinone (MEBQ). MEA may be oxidized by the P450 monooxygenase system to 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA), which can be hydrolytically spontaneously deaminated to MEBQ or MEHQ. The MEA microbial metabolic pathway was reconstituted based on the substrate spectra and identification of the intermediate metabolites in both the wild-type and mutant strains. Plasmidome sequencing indicated that both strains harbored 7 plasmids with sizes ranging from 6,108 bp to 287,745 bp. Among the 7 plasmids, 6 were identical, and pMEA02' in strain MEA3-1Mut lost a 37,000-bp fragment compared to pMEA02 in strain MEA3-1. Two-dimensional electrophoresis (2-DE) and protein mass fingerprinting (PMF) showed that MEA3-1Mut lost the two-component flavin-dependent monooxygenase (TC-FDM) MeaBA, which was encoded by a gene in the lost fragment of pMEA02. MeaA shared 22% to 25% amino acid sequence identity with oxygenase components of some TC-FDMs, whereas MeaB showed no sequence identity with the reductase components of those TC-FDMs. Complementation with meaBA in MEA3-1Mut and heterologous expression in Pseudomonas putida strain KT2440 resulted in the production of an active MEHQ monooxygenase.

Targeted inhibition of galectin 1 by thiodigalactoside dramatically reduces body weight gain in diet-induced obese rats.

Galectin 1 (GAL1), an animal lectin is well characterized in the context of cancer, tumor environment, but its physiological roles in obesity remain to be demonstrated. In this study, we investigated whether targeted inhibition of GAL1 prevents obesity based on the previous observations that GAL1 is highly expressed in adipose tissues of high-fat diet (HFD)-induced obese rats. Lipogenic capacity of Lgals1 knocked down adipocytes was evaluated by determining the expression levels of major lipogenic markers using real-time PCR and immunoblot analysis. GAL1 partner proteins were identified using co-immunoprecipitation followed by protein mass fingerprinting. Finally, inhibitory effect of GAL1 by thiodigalactoside (TDG) was assessed in adipocytes and HFD-induced obese rats. Knockdown of GAL1-encoding gene (Lgals1) attenuated adipogenesis and lipogenesis in both 3T3-L1 and HIB1B adipocytes. Further, direct treatment with TDG, a potent inhibitor of GAL1, to cultured adipocytes in vitro significantly reduced fat accumulation. Our animal experiment revealed that intraperitoneal injection of TDG (5 mg kg(-1)) once per week for 5 weeks in Sprague-Dawley (SD) rats resulted in dramatic inhibition of HFD-induced body weight gain (27.3% reduction compared with HFD-fed controls) by inhibiting adipogenesis and lipogensis as well as by increasing expression of the proteins associated with thermogenesis and energy expenditure. GAL1 has an essential role in HFD-induced obesity development. From a clinical viewpoint, pharmaceutical targeting of GAL1 using TDG and other inhibitor compounds would be a novel therapeutic approach for the treatment of obesity.

Feasibility of protein fingerprinting technology for detecting Tribolium castaneum (Herbst) insect fragments in wheat flour

The red flour beetle, Tribolium castaneum (Herbst), is one of the predominant insects that cause serious decrease in quality of wheat flour. Protein mass fingerprinting has the potential to accurately detect insect parts in wheat flour, based on special peptides of T. castaneum. For determining the capability of protein fingerprinting for detecting insect parts in wheat flour, T. castaneum, wheat flour, and mixtures of T. castaneum and wheat flour were tested in this study. Three protein extraction buffers: 50% acetonitrile; sterilized water; and the buffer of 10 mM imidazole pH 7.0, 1 mM phenylthiourea, 1% Triton X-100 and protease inhibitor cocktail (PTU buffer), were compared for insect protein detection. An Agilent 1100 reversed phase high performance liquid chromatography (RP-HPLC) instrument was used for peptide purification and fractionation. Matrix-assisted laser desorption/ionization (MALDI) was used to read the RP-HPLC fractions, and the results were analyzed using sMART Engine, details given later. Fifty percent acetonitrile was adopted as protein extract buffer. Since very limited information about T. castaneum protein can be found in the database of sMART and National Center for Biotechnology Information (NCBI), peptide mapping method was used for identifying insect from the wheat flour. Peptide mapping method, which could set up biomarkers for each type of insect, was proven to be a more effective and practical way to detect insect contamination. This peptide mapping technique could obtain 78 biomarkers of T. castaneum, when T. castaneum was 0.1% in wheat flour by mass. This sensitivity needs to be increased further during future research.

Identification of field-caught Culicoides biting midges using matrix-assisted laser desorption/ionization time of flight mass spectrometry

Culicoides biting midges are of great importance as vectors of pathogens and elicitors of allergy. As an alternative for the identification of these tiny insects, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was evaluated. Protein mass fingerprints were determined for 4-5 field-caught reference (genetically confirmed) individuals of 12 Culicoides species from Switzerland, C. imicola from France, laboratory-reared C. nubeculosus and a non-biting midge. Reproducibility and accuracy of the database was tested in a validation study by analysing 108 mostly field-caught target Culicoides midges and 3 specimens from a non-target species. A reference database of biomarker mass sets containing between 24 and 38 masses for the different species could be established. Automated database-based identification was achieved for 101 of the 108 specimens. The remaining 7 midges required manual full comparison with the reference spectra yielding correct identification for 6 specimens and an ambiguous result for the seventh individual. Specimens of the non-target species did not yield identification. Protein profiling by MALDI-TOF, which is compatible with morphological and genetic identification of specimens, can be used as an alternative, quick and inexpensive tool to accurately identify Culicoides biting midges collected in the field.

Basic mechanisms of Friend virus-induced immunomodulation and tumorigenesis (154.34)

Abstract Friend murine leukemia Virus (FV) infection of immunocompetent mice is a well established model to acquire further knowledge about retrovirus-associated mechanisms, enabling the development of preventive and therapeutic treatments against retrovirus-induced diseases. Our own studies show that formation of the immunological synapse between FV-infected DCs and T cells is altered. Those DCs induced a regulatory phenotype and suppressive function in cocultured CD4+ T cells (Treg). In order to identify key molecules that contribute to this Treg induction, expression patterns of cytoplasmatic proteins in uninfected and FV-infected DCs were comparatively analyzed by Protein-mass fingerprinting. By that approach about 600 differentially expressed proteins were identified. Interestingly, many of these are cytoskeletal proteins, and some are known to be involved in DC-T cell interactions (e.g. Myosins1F, 9, and 14). Notably, several MHCII antigens are downregulated in FV-infected DCs as compared with uninfected DCs. Recent studies show that the endocytic capacity of FV-infected DCs was significantly enhanced as compared with control DCs. Our current work is focussed on elucidating the function of signalling proteins which are differentially expressed in FV-infected vs. uninfected DCs. Taken together, our work is aimed to understand how retroviral infection of DCs alters DC-T cell interactions, and at which extent these effects contribute to retrovirus-induced immunosuppression.

Rapid identification of Legionella spp. by MALDI-TOF MS based protein mass fingerprinting

A set of reference strains representing 38 different Legionella species were submitted to Whole Cell Mass Spectrometry (WCMS) with MALDI-TOF. The dendrogram computed from strain mass spectral patterns obtained by WCMS was compared to the phylogenetic tree obtained from macrophage infectivity potentiator ( mip) sequences. The trees inferred from these two methods revealed significant homologies. Using 453 Legionella isolates previously characterized by genotyping, it was possible to create species-specific SuperSpectra, using appropriate sets of spectral masses, allowing unambiguous differentiation and identification of the most frequently isolated Legionella species. These SuperSpectra were tested for their suitability to identify Legionella strains isolated from water samples, cooling towers, potting soils and patient specimens deposited at the Swiss National Reference Centre for Legionella and previously identified by molecular methods such as mip gene sequencing. 99.1% of the tested strains isolated from the environment could be correctly identified by comparison with the new SuperSpectra. The identification of Legionella spp. by MALDI-TOF MS is rapid, easy to perform and has the advantage of being time- and cost-saving, in comparison to sequence-based identification.

ATP-binding cassette transporter isoform C2 localizes to the apical plasma membrane via interactions with scaffolding protein

ATP-binding cassette transporter isoform C2 (ABCC2) localizes to the apical plasma membrane in polarized cells. Apical localization of ABCC2 in hepatocytes plays an important role in biliary excretion of endobiotics and xenobiotics, but the mechanism by which ABCC2 localizes to the apical membrane has not been conclusively elucidated. Here, we investigate the role of scaffolding proteins on ABCC2 localization with a focus on the function of PDZK1 (post-synaptic density 95/disk large/zonula occludens-1 domain containing 1) in regulating ABCC2 localization. The C-terminal 77 residues of ABCC2 were used to probe interacting proteins from HepG2 cells. Protein mass fingerprinting identified PDZK1 as a major interacting protein. PDZK1 associated with the plasma membrane, most likely at the apical vacuoles of HepG2 cells. Affinity pull-down assays confirmed that the C-terminal NSTKF of ABCC2 bound to the fourth PDZ domain of PDZK1. Removal of this PDZ-binding motif significantly reduced the normal apical localization of ABCC2. In HepG2 cells, overexpression of this fourth domain overcame endogenous PDZK1 and reduced the ABCC2 localization at the apical membrane with a reciprocal increase of intracellular accumulation of mislocalized ABCC2. These results suggest a possible role for an interaction between ABCC2 and PDZK1 in apical localization of ABCC2 in hepatocytes.