Interrogating the Venom of the Viperid Snake Sistrurus catenatus edwardsii by a Combined Approach of Electrospray and MALDI Mass Spectrometry

The complete sequence characterization of snake venom proteins by mass spectrometry is rather challenging due to the presence of multiple isoforms from different protein families. In the present study, we investigated the tryptic digest of the venom of the viperid snake Sistrurus catenatus edwardsii by a combined approach of liquid chromatography coupled to either electrospray (online) or MALDI (offline) mass spectrometry. These different ionization techniques proved to be complementary allowing the identification a great variety of isoforms of diverse snake venom protein families, as evidenced by the detection of the corresponding unique peptides. For example, ten out of eleven predicted isoforms of serine proteinases of the venom of S. c. edwardsii were distinguished using this approach. Moreover, snake venom protein families not encountered in a previous transcriptome study of the venom gland of this snake were identified. In essence, our results support the notion that complementary ionization techniques of mass spectrometry allow for the detection of even subtle sequence differences of snake venom proteins, which is fundamental for future structure-function relationship and possible drug design studies

A systematic approach to identify STRE-binding proteins of the gsn glycogen synthase gene promoter in Neurospora crassa

The gene encoding glycogen synthase in Neurospora crassa (gsn) is transcriptionally down-regulated when mycelium is exposed to a heat shock from 30 to 45 degrees C. The gsn promoter has one stress response element (STRE) motif that is specifically bound by heat shock activated nuclear proteins. In this work, we used biochemical approaches together with mass spectrometric analysis to identify the proteins that bind to the STRE motif and could participate in the gsn transcription regulation during heat shock. Crude nuclear extract of heat-shocked mycelium was prepared and fractionated by affinity chromatography. The fractions exhibiting DNA-binding activity were identified by electrophoretic mobility shift assay (EMSA) using as probe a DNA fragment containing the STRE motif DNA-protein binding activity was confirmed by Southwestern analysis. The molecular mass (MM) of proteins was estimated by fractionating the crude nuclear extract by SDS-PAGE followed by EMSA analysis of the proteins corresponding to different MM intervals. Binding activity was detected at the 30-50 MM kDa interval. Fractionation of the crude nuclear proteins by IEF followed by EMSA analysis led to the identification of two active fractions belonging to the pIs intervals 3.54-4.08 and 6.77-7.31. The proteins comprising the MM and pI intervals previously identified were excised from a 2-DE gel, and subjected to mass spectrometric analysis (MALDI-TOF/TOF) after tryptic digestion. The proteins were identified by search against the MIPS and MIT N. crassa databases and five promising candidates were identified. Their structural characteristics and putative roles in the gsn transcription regulation are discussed

Proteomic Deep Mining the Venom of the Red-Headed Krait, Bungarus flaviceps

The use of -omics technologies allows for the characterization of snake venom composition at a fast rate and at high levels of detail. In the present study, we investigated the protein content of Red-headed Krait (Bungarus flaviceps) venom. This analysis revealed a high diversity of snake venom protein families, as evidenced by high-throughput mass spectrometric analysis. We found all six venom protein families previously reported in a transcriptome study of the venom gland of B. flaviceps, including phospholipases A2 (PLA2s), Kunitz-type serine proteinase inhibitors (KSPIs), three-finger toxins (3FTxs), cysteine-rich secretory proteins (CRISPs), snaclecs, and natriuretic peptides. A combined approach of automated database searches and de novo sequencing of tandem mass spectra, followed by sequence similarity searches, revealed the presence of 12 additional toxin families. De novo sequencing alone was able to identify 58 additional peptides, and this approach contributed significantly to the comprehensive description of the venom. Abundant protein families comprise 3FTxs (22.3%), KSPIs (19%), acetylcholinesterases (12.6%), PLA2s (11.9%), venom endothelial growth factors (VEGFs, 8.4%), nucleotidases (4.3%), and C-type lectin-like proteins (snaclecs, 3.3%); an additional 11 toxin families are present at significantly lower concentrations, including complement depleting factors, a family not previously detected in Bungarus venoms. The utility of a multifaceted approach toward unraveling the proteome of snake venoms, employed here, allowed detection of even minor venom components. This more in-depth knowledge of the composition of B. flaviceps venom facilitates a better understanding of snake venom molecular evolution, in turn contributing to more effective treatment of krait bites

Comparative proteomics of cerebrospinal fluid reveals a predictive model for differential diagnosis of pneumococcal, meningococcal, and enteroviral meningitis, and novel putative therapeutic targets

Submitted by sandra infurna ([email protected]) on 2016-03-10T14:47:14Z No. of bitstreams: 1 alex_chapeaurouge_etal_IOC_2015.pdf: 343462 bytes, checksum: deaf5a5bf883dc052b8cdda7882ad5b1 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-03-10T15:07:20Z (GMT) No. of bitstreams: 1 alex_chapeaurouge_etal_IOC_2015.pdf: 343462 bytes, checksum: deaf5a5bf883dc052b8cdda7882ad5b1 (MD5)Made available in DSpace on 2016-03-10T15:07:20Z (GMT). No. of bitstreams: 1 alex_chapeaurouge_etal_IOC_2015.pdf: 343462 bytes, checksum: deaf5a5bf883dc052b8cdda7882ad5b1 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou (CPqRR). Grupo de Pesquisa em Genômica e Biologia Computacional. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou (CPqRR). Grupo de Pesquisa em Genômica e Biologia Computacional. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou (CPqRR). Grupo de Pesquisa em Genômica e Biologia Computacional. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou (CPqRR). Grupo de Pesquisa em Genômica e Biologia Computacional. Belo Horizonte, MG, Brasil.FHEMIG. Hospital de Crianças João Paulo II. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou (CPqRR). Grupo de Pesquisa Informática de Biosistemas. Belo Horizonte, MG, Brasil.Background: Meningitis is the inflammation of the meninges in response to infection or chemical agents. While aseptic meningitis, most frequently caused by enteroviruses, is usually benign with a self-limiting course, bacterial meningitis remains associated with high morbidity and mortality rates, despite advances in antimicrobial therapy and intensive care. Fast and accurate differential diagnosis is crucial for assertive choice of the appropriate therapeutic approach for each form of meningitis. Methods: We used 2D-PAGE and mass spectrometry to identify the cerebrospinal fluid proteome specifically related to the host response to pneumococcal, meningococcal, and enteroviral meningitis. The disease-specific proteome signatures were inspected by pathway analysis. Results: Unique cerebrospinal fluid proteome signatures were found to the three aetiological forms of meningitis investigated, and a qualitative predictive model with four protein markers was developed for the differential diagnosis of these diseases. Nevertheless, pathway analysis of the disease-specific proteomes unveiled that Kallikrein-kinin system may play a crucial role in the pathophysiological mechanisms leading to brain damage in bacterial meningitis. Proteins taking part in this cellular process are proposed as putative targets to novel adjunctive therapies. Conclusions: Comparative proteomics of cerebrospinal fluid disclosed candidate biomarkers, which were combined in a qualitative and sequential predictive model with potential to improve the differential diagnosis of pneumococcal, meningococcal and enteroviral meningitis. Moreover, we present the first evidence of the possible implication of Kallikrein-kinin system in the pathophysiology of bacterial meningitis

Identification of immunogenic proteins of the bacterium Acinetobacter baumannii using a proteomic approach

Made available in DSpace on 2015-06-08T14:01:53Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) anapaula_assefetal_IOC_2014.pdf: 1859957 bytes, checksum: 09c3b5c760f35acea9aafe7444daa579 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil.undação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.undação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa de Infecções Hospitalares. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia de Imunobiológicos. Rio de Janeiro, RJ, Brasil.Purpose: Acinetobacter baumannii is an important opportunistic pathogen that causes pneumoniae, urinary tract infections, and/or septicemia in immunocompromised patients. This pathogen is frequently associated with nosocomial outbreaks worldwide and has become particularly problematic because of its prevalence and resistance patterns to several antibiotics. In the present study, we used an immunoproteome-based approach to identify immunogenic proteins located on the surface of A. baumannii for the development of a possible immunotherapy against this devastating bacterial infection. Experimental design: Sera from patients with A. baumannii infections (n = 50) and from a control group of healthy individuals (n = 3) were analyzed for reactivity against A. baumannii outer membrane proteins (OMPs) using Western blot analysis. To identify potential immunogenic proteins in A. baumannii,OMPs were separated by 2DE (2Delectrophoresis), and reactive sera from infected patients were randomly selected and divided into two different pools, each containing 15 sera. Finally, MALDI-TOF/TOF mass spectrometric analysis was employed to identify the corresponding proteins. Results: This analysis identified six immunoreactive proteins: OmpA, Omp34kDa, OprC, OprB-like, OXA-23, and ferric siderophore receptor protein. Notably, these proteins are highly abundant on the bacterial surface and involved in virulence, antibiotic resistance, and growth. Conclusions and clinical relevance: Our results support the notion that the proteins identified in the present immunoproteome study could serve as antigen candidates for the development of vaccines and passive immunotherapies against A. baumannii infections

Sequence coverages of some of the predicted venom gland proteins of <i>S</i>. <i>c</i>. <i>edwardsii</i> as revealed by the combined approach of MALDI and ESI tandem mass spectrometry.

<p>Protein sequences including specific domains are indicted by colored bars; below these, corresponding peptides identified by ESI (black lines) and MALDI (red lines) are indicated.</p

Sequence alignment of the metalloproteinases of the venom of <i>S</i>. <i>c</i>. <i>edwardsii</i>.

<p>Different colors indicate the unique peptides identified by tandem mass spectrometry of the corresponding proteins.</p

Sequence alignment of the serine proteinases of the venom of <i>S</i>. <i>c</i>. <i>edwardsii</i>.

<p>Different colors indicate the unique peptides identified by tandem mass spectrometry of the corresponding proteins.</p

Identification of proteins of the venom of <i>S</i>. <i>c</i>. <i>edwardsii</i> not predicted from the transcriptome analysis.

<p>Identification of proteins of the venom of <i>S</i>. <i>c</i>. <i>edwardsii</i> not predicted from the transcriptome analysis.</p