scholarly journals Purificação e avaliação da interferência de BthMP (uma metaloprotease da peçonha de Bothrops moojeni) na coagulação sanguínea

2016 ◽  
Vol 1 (2) ◽  
pp. 7
Author(s):  
Manuela Andrade Santos ◽  
Luzia Aparecida Pando ◽  
Veridiana De Melo Rodrigues ◽  
Mariana De Souza Castro ◽  
Mário Sérgio Rocha Rocha Gomes
Keyword(s):  
Peptide Mass Fingerprinting ◽  
Maldi Tof ◽  
Peptide Mass ◽  
Sds Page ◽  
Bothrops Moojeni

Neste trabalho relatamos a purificação da metaloprotease BthMP, proveniente da peçonha da serpente Bothrops moojeni. Para a purificação desta protease, utilizaram-se os passos cromatográficos de troca iônica (DEAE-Sepharose) e de exclusão molecular (Sephadex G-75), sendo o produto desses processos uma banda proteica com elevado grau de pureza, visualizada em SDS-PAGE a 14%, denominada BthMP. Esta, por sua vez, quando analisada em MALDI-TOF revelou a massa molecular nativa de 23.050 Da e 23.872 Da na forma reduzida, e a partir dos fragmentos peptídicos obtidos por Peptide Mass Fingerprinting (PMF) em MS (MALDI-TOF/TOF) indicou alta similaridade com a metaloprotease BmooMPα-I. Em termos enzimáticos, BthMP mostrou atividade proteolítica sobre azocaseína e frente ao PMSF e benzamidina, enquanto que esta atividade foi inibida na presença de EDTA, 1,10-fenantrolina e β-mercaptoetanol, sendo portanto uma metaloprotease zinco dependente da classe P-I. Ainda com este propósito, verificou-se sua especificidade enzimática sobre as cadeias Aα e Bβ do fibrinogênio, e também o consumo de fibrinogênio in vivo. Foi constatado ainda sua ação em componentes da cascata de coagulação, devido ao prolongamento do Tempo de Protrombina (TP) e do Tempo de Tromboplastina Parcial ativada (TTPa). Desta forma, a acentuada atividade fibrinogenolítica e o alto consumo de fibrinogênio in vivo são resultados que indicam a ação anticoagulante da BthMP; além do mais, sua capacidade de interferir na cascata de coagulação sugere que esta protease é promissora para futuros estudos que possam indicar um novo modelo de fármaco antitrombótico. https://doi.galoa.com.br/doi/10.17648/jibi-2448-0002-1-2-5128

Purificação e avaliação da interferência de BthMP (uma metaloprotease da peçonha de Bothrops moojeni) na coagulação sanguínea

2016 ◽  
Vol 1 (2) ◽  
Author(s):  
Manuela Andrade Santos ◽  
Luzia Aparecida Pando ◽  
Veridiana de Melo Rodrigues ◽  
Mariana de Souza Castro ◽  
Mário Sérgio Rocha Gomes
Keyword(s):  
Peptide Mass Fingerprinting ◽  
Maldi Tof ◽  
Peptide Mass ◽  
Sds Page ◽  
Bothrops Moojeni

Neste trabalho relatamos a purificação da metaloprotease BthMP, proveniente da peçonha da serpente Bothrops moojeni. Para a purificação desta protease, utilizaram-se os passos cromatográficos de troca iônica (DEAE-Sepharose) e de exclusão molecular (Sephadex G-75), sendo o produto desses processos uma banda proteica com elevado grau de pureza, visualizada em SDS-PAGE a 14%, denominada BthMP. Esta, por sua vez, quando analisada em MALDI-TOF revelou a massa molecular nativa de 23.050 Da e 23.872 Da na forma reduzida, e a partir dos fragmentos peptídicos obtidos por Peptide Mass Fingerprinting (PMF) em MS (MALDI-TOF/TOF) indicou alta similaridade com a metaloprotease BmooMPα-I. Em termos enzimáticos, BthMP mostrou atividade proteolítica sobre azocaseína e frente ao PMSF e benzamidina, enquanto que esta atividade foi inibida na presença de EDTA, 1,10-fenantrolina e β-mercaptoetanol, sendo portanto uma metaloprotease zinco dependente da classe P-I. Ainda com este propósito, verificou-se sua especificidade enzimática sobre as cadeias Aα e Bβ do fibrinogênio, e também o consumo de fibrinogênio in vivo. Foi constatado ainda sua ação em componentes da cascata de coagulação, devido ao prolongamento do Tempo de Protrombina (TP) e do Tempo de Tromboplastina Parcial ativada (TTPa). Desta forma, a acentuada atividade fibrinogenolítica e o alto consumo de fibrinogênio in vivo são resultados que indicam a ação anticoagulante da BthMP; além do mais, sua capacidade de interferir na cascata de coagulação sugere que esta protease é promissora para futuros estudos que possam indicar um novo modelo de fármaco antitrombótico.

scholarly journals Stable isotope labelling in vivo as an aid to protein identification in peptide mass fingerprinting

PROTEOMICS ◽  
2002 ◽  
Vol 2 (2) ◽  
pp. 157-163 ◽  
Author(s):  
Julie M. Pratt ◽  
Duncan H. L. Robertson ◽  
Simon J. Gaskell ◽  
Isabel Riba-Garcia ◽  
Simon J. Hubbard ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Protein Identification ◽  
Peptide Mass Fingerprinting ◽  
Stable Isotope Labelling ◽  
Isotope Labelling ◽  
Peptide Mass

scholarly journals Site-Specific N-Glycosylation on the AAV8 Capsid Protein

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 644 ◽  
Author(s):  
Arya Aloor ◽  
Junping Zhang ◽  
Ebtesam Gashash ◽  
Aishwarya Parameswaran ◽  
Matthew Chrzanowski ◽  
...  
Keyword(s):  
Capsid Protein ◽  
High Resolution Mass Spectrometry ◽  
Genetic Diseases ◽  
Peptide Mass Fingerprinting ◽  
Gene Therapy Vector ◽  
Host Cell Proteins ◽  
Peptide Mass ◽  
293 Cells ◽  
Proteome Discoverer

Adeno associated virus (AAV) is a versatile gene delivery tool, which has been approved as a human gene therapy vector for combating genetic diseases. AAV capsid proteins are the major components that determine the tissue specificity, immunogenicity and in vivo transduction performance of the vector. In this study, the AAV8 capsid glycosylation profile was systemically analyzed by peptide mass fingerprinting utilizing high-resolution mass spectrometry to determine the presence of capsid glycosylation. We identified N-glycosylation on the amino acid N499 of the capsid protein. We characterized the overall sugar profile for vector produced in 293 cells. Multiple N-glycosylated host-cell proteins (HCPs) copurified with AAV8 vectors and were identified by analyzing LC-MS data utilizing a human database and proteome discoverer search engine. The N-glycosylation analysis by MALDI-TOF MS, highlighted the probability of AAV8 interaction with terminal galactosylated N-glycans within the HCPs.

scholarly journals Altered Protein Expression of Streptococcus oralis Cultured at Low pH Revealed by Two-Dimensional Gel Electrophoresis

2001 ◽  
Vol 67 (8) ◽  
pp. 3396-3405 ◽  
Author(s):  
Joanna C. Wilkins ◽  
Karen A. Homer ◽  
David Beighton
Keyword(s):  
Gel Electrophoresis ◽  
Peptide Mass Fingerprinting ◽  
Low Ph ◽  
Ionization Mass ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Altered Expression ◽  
Streptococcus Oralis ◽  
Peptide Mass

ABSTRACT Streptococcus oralis is the predominant aciduric nonmutans streptococcus isolated from the human dentition, but the role of this organism in the initiation and progression of dental caries has yet to be established. To identify proteins that are differentially expressed by S. oralis growing under conditions of low pH, soluble cellular proteins extracted from bacteria grown in batch culture at pH 5.2 or 7.0 were analyzed by two-dimensional (2-D) gel electrophoresis. Thirty-nine proteins had altered expression at low pH; these were excised, digested with trypsin using an in-gel protocol, and further analyzed by peptide mass fingerprinting using matrix-assisted laser desorption ionization mass spectrometry. The resulting fingerprints were compared with the genomic database forStreptococcus pneumoniae, an organism that is phylogenetically closely related to S. oralis, and putative functions for the majority of these proteins were determined on the basis of functional homology. Twenty-eight proteins were up-regulated following growth at pH 5.2; these included enzymes of the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase), the polypeptide chains comprising ATP synthase, and proteins that are considered to play a role in the general stress response of bacteria, including the 60-kDa chaperone, Hsp33, and superoxide dismutase, and three distinct ABC transporters. These data identify, for the first time, gene products that may be important in the survival and proliferation of nonmutans aciduric S. oralis under conditions of low pH that are likely to be encountered by this organism in vivo.

scholarly journals Genetic and Functional Analysis of the Soluble Oxaloacetate Decarboxylase from Corynebacterium glutamicum

2010 ◽  
Vol 192 (10) ◽  
pp. 2604-2612 ◽  
Author(s):  
Simon Klaffl ◽  
Bernhard J. Eikmanns
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Peptide Mass Fingerprinting ◽  
Transcriptional Analysis ◽  
Lysine Production ◽  
Ionization Time ◽  
Peptide Mass ◽  
Methyltransferase Gene ◽  
High Level

ABSTRACT Soluble, divalent cation-dependent oxaloacetate decarboxylases (ODx) catalyze the irreversible decarboxylation of oxaloacetate to pyruvate and CO2. Although these enzymes have been characterized in different microorganisms, the genes that encode them have not been identified, and their functions have been only poorly analyzed so far. In this study, we purified a soluble ODx from wild-type C. glutamicum about 65-fold and used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis and peptide mass fingerprinting for identification of the corresponding odx gene. Inactivation and overexpression of odx led to an absence of ODx activity and to a 30-fold increase in ODx specific activity, respectively; these findings unequivocally confirmed that this gene encodes a soluble ODx. Transcriptional analysis of odx indicated that there is a leaderless transcript that is organized in an operon together with a putative S-adenosylmethionine-dependent methyltransferase gene. Biochemical analysis of ODx revealed that the molecular mass of the native enzyme is about 62 ± 1 kDa and that the enzyme is composed of two ∼29-kDa homodimeric subunits and has a Km for oxaloacetate of 1.4 mM and a V max of 201 μmol of oxaloacetate converted per min per mg of protein, resulting in a k cat of 104 s−1. Introduction of plasmid-borne odx into a pyruvate kinase-deficient C. glutamicum strain restored growth of this mutant on acetate, indicating that a high level of ODx activity redirects the carbon flux from oxaloacetate to pyruvate in vivo. Consistently, overexpression of the odx gene in an l-lysine-producing strain of C. glutamicum led to accumulation of less l-lysine. However, inactivation of the odx gene did not improve l-lysine production under the conditions tested.

scholarly journals Rapid, Sensitive, and Specific Escherichia coli H Antigen Typing by Matrix-Assisted Laser Desorption Ionization–Time of Flight-Based Peptide Mass Fingerprinting

2015 ◽  
Vol 53 (8) ◽  
pp. 2480-2485 ◽  
Author(s):  
Huixia Chui ◽  
Michael Chan ◽  
Drexler Hernandez ◽  
Patrick Chong ◽  
Stuart McCorrister ◽  
...  
Keyword(s):  
Peptide Mass Fingerprinting ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Content Type ◽  
Maldi Tof Ms ◽  
E Coli ◽  
Maldi Tof ◽  
Peptide Mass ◽  
Reference Strains ◽  
Tof Ms

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has gained popularity in recent years for rapid bacterial identification, mostly at the genus or species level. In this study, a rapid method to identify the Escherichia coli flagellar antigen (H antigen) at the subspecies level was developed using a MALDI-TOF MS platform with high specificity and sensitivity. Flagella were trapped on a filter membrane, and on-filter trypsin digestion was performed. The tryptic digests of each flagellin then were collected and analyzed by MALDI-TOF MS through peptide mass fingerprinting. Sixty-one reference strains containing all 53 H types and 85 clinical strains were tested and compared to serotyping designations. Whole-genome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix for MALDI-TOF MS, with higher confidence during protein identification. After method optimization, reference strains representing all 53 E. coli H types were identified correctly by MALDI-TOF MS. A custom E. coli flagellar/H antigen database was crucial for clearly identifying the E. coli H antigens. Of 85 clinical isolates tested by MALDI-TOF MS-H, 75 identified MS-H types (88.2%) matched results obtained from traditional serotyping. Among 10 isolates where the results of MALDI-TOF MS-H and serotyping did not agree, 60% of H types characterized by whole-genome sequencing agreed with those identified by MALDI-TOF MS-H, compared to only 20% by serotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coli H antigen identification, especially during E. coli outbreaks.

scholarly journals Isethionate formation from taurine in Chromohalobacter salexigens: purification of sulfoacetaldehyde reductase

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1547-1555 ◽  
Author(s):  
Zdeněk Krejčík ◽  
Klaus Hollemeyer ◽  
Theo H. M. Smits ◽  
Alasdair M. Cook
Keyword(s):  
Sequence Data ◽  
Gene Clusters ◽  
Peptide Mass Fingerprinting ◽  
Rt Pcr ◽  
Regulatory Enzymes ◽  
Peptide Mass ◽  
Sds Page ◽  
Short Chain Alcohol ◽  
Chromohalobacter Salexigens ◽  
Similar Gene

Bacterial generation of isethionate (2-hydroxyethanesulfonate) from taurine (2-aminoethanesulfonate) by anaerobic gut bacteria was established in 1980. That phenomenon in pure culture was recognized as a pathway of assimilation of taurine-nitrogen. Based on the latter work, we predicted from genome-sequence data that the marine gammaproteobacterium Chromohalobacter salexigens DSM 3043 would exhibit this trait. Quantitative conversion of taurine to isethionate, identified by mass spectrometry, was confirmed, and the taurine-nitrogen was recovered as cell material. An eight-gene cluster was predicted to encode the inducible vectorial, scalar and regulatory enzymes involved, some of which were known from other taurine pathways. The genes (Csal_0153–Csal_0156) encoding a putative ATP-binding-cassette (ABC) transporter for taurine (TauAB1B2C) were shown to be inducibly transcribed by reverse transcription (RT-) PCR. An inducible taurine : 2-oxoglutarate aminotransferase [EC 2.6.1.55] was found (Csal_0158); the reaction yielded glutamate and sulfoacetaldehyde. The sulfoacetaldehyde was reduced to isethionate by NADPH-dependent sulfoacetaldehyde reductase (IsfD), a member of the short-chain alcohol dehydrogenase superfamily. The 27 kDa protein (SDS-PAGE) was identified by peptide-mass fingerprinting as the gene product of Csal_0161. The putative exporter of isethionate (IsfE) is encoded by Csal_0160; isfE was inducibly transcribed (RT-PCR). The presumed transcriptional regulator, TauR (Csal_0157), may autoregulate its own expression, typical of GntR-type regulators. Similar gene clusters were found in several marine and terrestrial gammaproteobacteria, which, in the gut canal, could be the source of not only mammalian, but also arachnid and cephalopod isethionate.

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