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Yulanda M Williamson, Hercules Moura,
Kaneatra Simmons,
Jennifer Whitmon,
Nikkol Melnick,
Jon Rees,
Adrian Woolfitt,
David M Schieltz,
Maria L Tondella,
Edwin Ades,
Jacquelyn Sampson,
George Carlone,
John R Barr
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ABSTRACT: Bordetella pertussis (Bp) is the etiologic agent of pertussis or whooping cough, a highly contagious respiratory disease occurring primarily in infants and young children. Although vaccine preventable, pertussis cases have increased over the years leading researchers to re-evaluate vaccine control strategies. Since bacterial outer membrane proteins, comprising the surfaceome, often play roles in pathogenesis and antibody-mediated immunity, three recent Bp circulating isolates were examined using proteomics to identify any potential changes in surface protein expression. Fractions enriched for outer membrane proteins were digested with trypsin and the peptides analyzed by nano liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS), followed by database analysis to elucidate the surfaceomes of our three Bp isolates. Furthermore, a less labor intensive non-gel based antibody affinity capture technology in conjunction with MS was employed to assess each Bp strains' immunogenic outer membrane proteins. This novel technique is generally applicable allowing for the identification of immunogenic surface expressed proteins on pertussis and other pathogenic bacteria.
Journal of microbiological methods 04/2012; 90(2):119-33. · 2.43 Impact Factor
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Rolieria West,
Jennifer Whitmon,
Yulanda M Williamson, Hercules Moura,
Marguerite Nelson,
Nikkol Melnick,
Maria Lucia C Tondella,
David Schieltz,
Jon Rees,
Adrian R Woolfitt,
John R Barr,
Edwin W Ades,
George M Carlone,
Jacquelyn S Sampson
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ABSTRACT: Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (>95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.
Journal of proteomics 03/2012; 75(6):1966-72. · 5.07 Impact Factor
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ABSTRACT: An immunocapture isotope dilution mass spectrometry (IC-IDMS) method was developed to quantify antibody-bound influenza hemagglutinins (HA) in trivalent influenza vaccines (TIV). Currently, regulatory potency requirements for TIV require HA quantification based on the single radial immunodiffusion (SRID) assay, which is time-consuming, laborious, and requires production of large quantities of reagents globally. In IC-IDMS, antiserum to the HA of interest captured viral proteins that were in the correct conformation to be recognized by the antibodies. The captured proteins were digested, and evolutionarily conserved tryptic peptides were quantified using isotope-dilution liquid chromatography-tandem mass spectrometry. IC-IDMS relies on antibody-antigen binding similar to SRID but incorporates the accuracy and precision of IDMS. Polyclonal antibodies (pAb-H3) prepared by injection of sheep with purified H3 HA captured 82.9% (55.26 fmol/μL) of the total H3 HA (66.69 fmol/μL) from the commercial TIV and 93.6% (57.23 fmol/μL) of the total H3 HA (61.14 fmol/μL) in purified virus. While other HA (H1, B), neuraminidase (N1, N2, NB), viral matrix proteins, and nucleoproteins were also captured by this antiserum, our results were not affected due to the specificity of the mass spectrometer. IC-IDMS is an accurate, precise, sensitive, and selective method to measure antibody-bound HA in purified virus and commercial vaccines.
Analytical Chemistry 06/2011; 83(12):4729-37. · 5.86 Impact Factor
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ABSTRACT: There have been several outbreaks of botulism among poultry and wild birds in Sweden in recent years. The National Veterinary Institute of Sweden (SVA) has identified botulinum neurotoxin (BoNT)/C1 or the mosaic BoNT/C1D using the mouse bioassay. This is believed to be the first report on the application of the Endopep mass spectrometry (Endopep-MS) method to selected clinical animal (serum and liver) samples and a feed sample that had previously given positive test results with the mouse bioassay. In the mouse bioassay eight of the eleven samples were found to be neutralized by both BoNT/C1 and /D antitoxins; the other three were neutralized only by BoNT/C1 antitoxin, but the mice showed a prolonged survival time when the samples had been treated with /D antitoxin. The Endopep-MS analysis, on the other hand, demonstrated only BoNT/C1 activity for all eleven samples. This suggests that at least eight of the samples were of the chimeric toxin type BoNT/C1D, where the enzymically active site is identical to that of BoNT/C1, while other parts of the protein contain sequences of BoNT/D. This is the first step of a cross-validation between the established mouse bioassay and the Endopep-MS of serotypes BoNT/C1 and /C1D. Endopep-MS is concluded to have potential as an attractive alternative to the mouse bioassay.
Journal of Medical Microbiology 05/2011; 60(Pt 9):1299-305. · 2.50 Impact Factor
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ABSTRACT: Matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI) tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA) which combines with lethal factor (LF) and edema factor (EF), forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase.
Molecules 01/2011; 16(3):2391-413. · 2.39 Impact Factor
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ABSTRACT: Clostridium botulinum is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an in silico and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous literature to provide a complete description of the BoNT/G complex.
An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs--nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison.
The in silico protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of C. botulinum. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data-independent MS(E) data analysis, coupled to label-free quantification software, suggested that the weight ratio BoNT:NAPs is 1:3, whereas the molar ratio of BoNT:NTNH:HA70:HA17 is 1:1:2:1, within the BoNT/G progenitor toxin.
BMC Microbiology 01/2011; 11:232. · 3.04 Impact Factor
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ABSTRACT: A range of mass spectrometry-based techniques have been used to identify, characterize and differentiate Bacillus anthracis, both in culture for forensic applications and for diagnosis during infection. This range of techniques could usefully be
considered to exist as a continuum, based on the degrees of specificity involved. We show two examples here, a whole-organism
fingerprinting method and a high-specificity assay for one unique protein, anthrax lethal factor.
Keywords
Bacillus anthracis
-Anthrax-Anthrax toxin-Anthrax lethal factor-MALDI MS-Fingerprinting-Statistical analysis-Quantification
12/2010: pages 83-97;
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ABSTRACT: Botulinum neurotoxins (BoNTs) are very potent toxins and category A biological threat agents. BoNT serotypes /C1 and /D affect birds and mammals and can be potentially lethal to humans. We have previously described the usefulness of the Endopep-MS method to detect the activity of BoNT A through G. This report was followed by the application of the method to clinical samples. The activity of the BoNT serotypes associated with human disease (/A, /B, /E, and /F) was successfully detected. However, BoNT/C and /D require different conditions for fast substrate cleavage, and a comprehensive description of a method to study BoNT/C and /D has not yet been reported. This work describes a new, optimized version of the Endopep-MS method to detect BoNTs /C1 and /DC either spiked directly in 20 μL of reaction buffer or spiked in a larger volume of buffer and further extracted using antibody-coated magnetic beads. It was found that the incubation temperature at 42 °C was more effective for both toxin serotypes, but each toxin serotype has an optimum cleavage pH. Additionally, we describe for the first time a proteomics study using a fast trypsin digestion method and label-free quantification of these toxin serotypes.
FEMS Immunology & Medical Microbiology 12/2010; 61(3):288-300. · 2.44 Impact Factor
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Yulanda M Williamson, Hercules Moura,
David Schieltz,
Jon Rees,
Adrian R Woolfitt,
James L Pirkle,
Jacquelyn S Sampson,
Maria L Tondella,
Edwin Ades,
George Carlone,
John R Barr
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ABSTRACT: Bordetella pertussis (Bp) is the causative agent of pertussis, a vaccine preventable disease occurring primarily in children. In recent years, there has been increased reporting of pertussis. Current pertussis vaccines are acellular and consist of Bp proteins including the major virulence factor pertussis toxin (Ptx), a 5-subunit exotoxin. Variation in Ptx subunit amino acid (AA) sequence could possibly affect the immune response. A blind comparative mass spectrometric (MS) analysis of commercially available Ptx as well as the chemically modified toxoid (Ptxd) from licensed vaccines was performed to assess peptide sequence and AA coverage variability as well as relative amounts of Ptx subunits. Qualitatively, there are similarities among the various sources based on AA percent coverages and MS/MS fragmentation profiles. Additionally, based on a label-free mass spectrometry-based quantification method there is differential relative abundance of the subunits among the sources.
Journal of Biomedicine and Biotechnology 01/2010; 2010:942365. · 2.44 Impact Factor
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ABSTRACT: Glycoproteins from the total vesicular fluid of Taenia crassiceps (VF-Tc) were prepared using three different purification methods, consisting of ConA-lectin affinity chromatography (ConA-Tc), preparative electrophoresis (SDS-PAGE) (14 gp-Tc), and monoclonal antibody immunoaffinity chromatography (18/14-Tc). The complex composition represented by the VF-Tc and ConA-Tc antigens revealed peptides ranging from 101- to 14-kDa and from 92- to 12-kDa, respectively. Immunoblotting using lectins confirmed glucose/mannose (glc/man) residues in the 18- and 14-kDa peptides, which are considered specific and immunodominant for the diagnosis of cysticercosis, and indicated that these fractions are glycoproteins. Serum antibodies from a patient with neurocysticercosis that reacted to the 14 gp band from T. crassiceps (Tc) were eluted from immunoblotting membranes and showed reactivity to 14 gp from Taenia solium. In order to determine the similar peptide sequence, the N-terminal amino acid was determined and analyzed with sequences available in public databases. This sequence revealed partial homology between T. crassiceps and T. solium peptides. In addition, mass spectrometry along with theoretical M(r) and pI of the 14 gp-Tc point suggested a close relationship to some peptides of a 150-kDa protein complex of the T. solium previously described. The identification of these common immunogenic sites will contribute to future efforts to develop recombinant antigens and synthetic peptides for immunological assays.
Experimental Parasitology 11/2009; 124(3):295-300. · 2.12 Impact Factor
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ABSTRACT: Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing death or respiratory failure leading to long-term intensive care. Treatment includes serotype-specific antitoxins, which must be administered early in the course of the intoxication. Rapidly determining human exposure to BoNT is an important public health goal. In previous work, our laboratory focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT/A-G serotypes in buffer and BoNT/A, /B, /E, and /F in clinical samples. We have previously reported the effectiveness of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. Because some antibodies inhibit or neutralize the activity of BoNT, the choice of antibody with which to extract the toxin is critical. In this work, we evaluated a panel of 16 anti-BoNT/A monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/A1, /A2, and /A3 complex as well as the recombinant LC of A1. We also evaluated the same antibody panel for the ability to extract BoNT/A1, /A2, and /A3. Among the mAbs, there were significant differences in extraction efficiency, ability to extract BoNT/A subtypes, and inhibitory effect on BoNT catalytic activity. The mAbs binding the C-terminal portion of the BoNT/A heavy chain had optimal properties for use in the Endopep-MS assay.
PLoS ONE 02/2009; 4(4):e5355. · 4.09 Impact Factor
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ABSTRACT: Streptococcus pneumoniae (pneumococcus [Pnc]) is a causative agent of many infectious diseases, including pneumonia, septicemia, otitis media, and conjunctivitis. There have been documented conjunctivitis outbreaks in which nontypeable (NT), nonencapsulated Pnc has been identified as the etiological agent. The use of mass spectrometry to comparatively and differentially analyze protein and peptide profiles of whole-cell microorganisms remains somewhat uncharted. In this report, we discuss a comparative proteomic analysis between NT S. pneumoniae conjunctivitis outbreak strains (cPnc) and other known typeable or NT pneumococcal and streptococcal isolates (including Pnc TIGR4 and R6, Streptococcus oralis, Streptococcus mitis, Streptococcus pseudopneumoniae, and Streptococcus pyogenes) and nonstreptococcal isolates (including Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus) as controls. cPnc cells and controls were grown to mid-log phase, harvested, and subsequently treated with a 10% trifluoroacetic acid-sinapinic acid matrix mixture. Protein and peptide fragments of the whole-cell bacterial isolate-matrix combinations ranging in size from 2 to 14 kDa were evaluated by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Additionally Random Forest analytical tools and dendrogramic representations (Genesis) suggested similarities and clustered the isolates into distinct clonal groups, respectively. Also, a peak list of protein and peptide masses was obtained and compared to a known Pnc protein mass library, in which a peptide common and unique to cPnc isolates was tentatively identified. Information gained from this study will lead to the identification and validation of proteins that are commonly and exclusively expressed in cPnc strains which could potentially be used as a biomarker in the rapid diagnosis of pneumococcal conjunctivitis.
Applied and environmental microbiology 09/2008; 74(19):5891-7. · 3.69 Impact Factor
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ABSTRACT: A novel mass spectral fingerprinting and proteomics approach using MALDI-TOF MS was applied to detect and identify protein biomarkers of group A Streptococcus (GAS) strains. Streptococcus pyogenes ATCC 700294 genome strain was compared with eight GAS clinical isolates to explore the ability of MALDI-TOF MS to differentiate isolates. Reference strains of other bacterial species were also analyzed and compared with the GAS isolates. MALDI preparations were optimized by varying solvents, matrices, plating techniques, and mass ranges for S. pyogenes ATCC 700294. Spectral variability was tested. A subset of common, characteristic, and reproducible biomarkers in the range of 2000-14 000 Da were detected, and they appeared to be independent of the culture media. Statistical analysis confirmed method reproducibility. Random Forest analysis of all selected GAS isolates revealed differences among most of them, and summed spectra were used for hierarchical cluster analysis. Specific biomarkers were found for each strain, and invasive GAS isolates could be differentiated. GAS isolates from cases of necrotizing fasciitis were clustered together and were distinct from isolates associated with noninvasive infections, despite their sharing the same emm type. Almost 30% of the biomarkers detected were tentatively identified as ribosomal proteins.
FEMS Immunology & Medical Microbiology 07/2008; 53(3):333-42. · 2.44 Impact Factor
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ABSTRACT: Among the many genera of free-living amoebae that exist in nature, members of only four genera have an association with human disease: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri and Sappinia diploidea. Acanthamoeba spp. and B. mandrillaris are opportunistic pathogens causing infections of the central nervous system, lungs, sinuses and skin, mostly in immunocompromised humans. Balamuthia is also associated with disease in immunocompetent children, and Acanthamoeba spp. cause a sight-threatening infection, Acanthamoeba keratitis, mostly in contact-lens wearers. Of more than 30 species of Naegleria, only one species, N. fowleri, causes an acute and fulminating meningoencephalitis in immunocompetent children and young adults. In addition to human infections, Acanthamoeba, Balamuthia and Naegleria can cause central nervous system infections in animals. Because only one human case of encephalitis caused by Sappinia diploidea is known, generalizations about the organism as an agent of disease are premature. In this review we summarize what is known of these free-living amoebae, focusing on their biology, ecology, types of disease and diagnostic methods. We also discuss the clinical profiles, mechanisms of pathogenesis, pathophysiology, immunology, antimicrobial sensitivity and molecular characteristics of these amoebae.
FEMS Immunology & Medical Microbiology 06/2007; 50(1):1-26. · 2.44 Impact Factor
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Journal of Eukaryotic Microbiology 05/2007; 44(s6):79s - 79s. · 2.66 Impact Factor
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ABSTRACT: Direct analysis in real time (DART) is implemented on a time-of-flight (TOF) mass spectrometer, and used for the generation of fatty acid methyl esters (FAMEs) ions from whole bacterial cells.
Chemical Communications 03/2007; · 6.17 Impact Factor
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ABSTRACT: Accurate bacterial identification is important in diagnosing disease and in microbial forensics. Coxiella burnetii, a highly infective microorganism causative of the human disease Q fever, is now considered a U.S. category B potential bioterrorism agent. We report here an approach for the confirmatory identification of C. burnetii at the strain level which involves the combined use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and supervised pattern recognition via Partial Least Squares-Discriminant Analysis (PLS-DA). C. burnetii isolates investigated in this study included the following prototype strains from different geographical and/or historical origins and with different antigenic properties: Nine Mile I, Australian QD, M44, KAV, PAV, Henzerling, and Ohio. After culture and purification following standard protocols, linear MALDI-TOF mass spectra of pure bacterial cultures were acquired in positive ion mode. Mass spectral data were normalized, baseline-corrected, denoised, binarized and modeled by PLS-DA under crossvalidation conditions. Robustness with respect to uncontrolled variations in the sample preparation and MALDI analysis protocol was assessed by repeating the experiment on five different days spanning a period of 6 months. The method was validated by the prediction of unknown C. burnetii samples in an independent test set with 100% sensitivity and specificity for five out of six strain classes.
Analytica chimica acta 02/2007; 583(1):23-31. · 4.31 Impact Factor
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ABSTRACT: Botulinum neurotoxin (BoNT) causes the disease botulism, which can be lethal if untreated. Previous work in our laboratory focused on developing Endopep-MS, a mass spectrometric-based endopeptidase method for the detection and differentiation of BoNT serotypes. We have expanded this effort to include an antibody capture method to partially purify and concentrate BoNT from serum and stool extract samples for the Endopep-MS assay. Because complex matrices such as serum and stool contain abundant endogenous proteases, this technique was needed to remove most proteases from the sample while concentrating BoNT from a sample size of 100 to 500 microl to 20 microl. When this antibody capture method is combined with the Endopep-MS reaction, limits of detection in 500mul of spiked human serum are 10 mouse LD50 (20 mouse LD50/ml) for BoNT A, 0.5 mouse LD50 (1 mouse LD50/ml) for BoNT B, 0.1 mouse LD50 (0.2 mouse LD50/ml) for BoNT E, and 0.5 mouse LD50 (1 mouse LD50/ml) for BoNT F. The limits of detection in spiked stool extracts are somewhat higher due to the high-protease environment of stool extract that also requires use of protease inhibitors. The entire method can be performed in as short a time as 4 h.
Analytical Biochemistry 05/2006; 351(1):84-92. · 3.00 Impact Factor
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ABSTRACT: HSA has been shown to react with many organic toxicants to form adducts that are useful biomarkers for exposure. Albumin isolation is an important first step for the analysis of these protein-toxicant adducts. We tested several approaches to isolate albumin from serum treated with an electrophilic organic toxicant known to form adducts with albumin, i.e., sulfur mustard agent (HD) (2,2'-dichloroethyl sulfide), in order to evaluate these techniques as purification methods. To select the most efficient isolation strategy, methods were evaluated using gel electrophoresis, total protein quantitation, and peptide-adduct identification by MS. Results suggest that the albumin-rich fractions obtained can be used to identify exposure by quantitating the albumin adducts to electrophilic organic toxicants such as HD. The HiTrap Blue HP albumin isolation system appears to display the most promising results for purifying albumin to detect HD-adducts, exhibiting high purification efficiency, satisfactory albumin recovery, promising specificity, and a higher loading capacity for serum.
PROTEOMICS 01/2006; 5(18):4973-9. · 4.51 Impact Factor
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ABSTRACT: Botulinum neurotoxins (BoNTs) are proteases that cleave specific cellular proteins essential for neurotransmitter release. Seven BoNT serotypes (A-G) exist; 4 usually cause human botulism (A, B, E, and F). We developed a rapid, mass spectrometry-based method (Endopep-MS) to detect and differentiate active BoNTs A, B, E, and F. This method uses the highly specific protease activity of the toxins with target peptides specific for each toxin serotype. The product peptides derived from the endopeptidase activities of BoNTs are detected by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. In buffer, this method can detect toxin equivalents of as little as 0.01 mouse lethal dose (MLD)50 and concentrations as low as 0.62 MLD50/mL. A high-performance liquid chromatography-tandem mass spectrometry method for quantifying active toxin, where the amount of toxin can be correlated to the amount of product peptides, is also described.
Emerging infectious diseases 11/2005; 11(10):1578-83. · 6.17 Impact Factor
