21 research outputs found
Proteomics Analyses of the Opportunistic Pathogen Burkholderia vietnamiensis Using Protein Fractionations and Mass Spectrometry
The main objectives of this work were to obtain a more extensive coverage of the Burkholderia vietnamiensis proteome than previously reported and to identify virulence factors using tandem mass spectrometry. The proteome of B. vietnamiensis was precipitated into four fractions to as extracellular, intracellular, cell surface and cell wall proteins. Two different approaches were used to analyze the proteins. The first was a gel-based method where 1D SDS-PAGE was used for separation of the proteins prior to reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS). The second method used MudPIT analysis (Multi dimensional Protein Identification Technique), where proteins are digested and separated using cation exchange and reversed phase separations before the MS/MS analysis (LC/LC-MS/MS). Overall, gel-based LC-MS/MS analysis resulted in more protein identifications than the MudPIT analysis. Combination of the results lead to identification of more than 1200 proteins, approximately 16% of the proteins coded from the annotated genome of Burkholderia species. Several virulence factors were detected including flagellin, porin, peroxiredoxin and zinc proteases
Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts
BACKGROUND: Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. RESULTS: Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. CONCLUSIONS: The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis
Identification of Residual Blood Proteins in Ticks by Mass Spectrometry Proteomics
Mass spectrometry–based proteomics of individual ticks demonstrated persistence of mammalian host blood components, including α- and β-globin chains, histones, and mitochondrial enzymes, in Ixodes scapularis and Amblyomma americanum ticks for months after molting. Residual host proteins may identify sources of infection for ticks
Identification of Residual Blood Proteins in Ticks by Mass Spectrometry Proteomics
Mass spectrometry–based proteomics of individual ticks demonstrated persistence of mammalian host blood components, including α- and β-globin chains, histones, and mitochondrial enzymes, in Ixodes scapularis and Amblyomma americanum ticks for months after molting. Residual host proteins may identify sources of infection for ticks
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A Multiplex Biomarker Approach for the Diagnosis of Transitional Cell Carcinoma from Canine Urine
Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. Commercial screening tests lack specificity and current diagnostic procedures are invasive. A proof of concept pilot project for analyzing the canine urinary proteome as a non-invasive diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of TCC, predicted the presence of disease with 90% accuracy.Keywords: Liquid chromatography, Biomarkers, Tandem mass spectrometry, Canine, Transitional cell carcinom
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Mass Spectometry Based Identification of Proteins in Burkholderia Species and in the Blood Meal of Ticks
Burkholderia pseudomallei is the causative agent of Melioidosis, an endemic disease in South East Asia, and is classified as a category B biological agent. Currently, there is no licensed vaccine for this disease; the mortality rate is high due to the incorrect diagnosis and the pathogen insusceptibility to general antibiotics. A mass spectrometry based proteomic approach has been applied in order to identify the proteins that are responsible for pathogenicity.Methods were developed for the proteomic analysis of Burkholderia species using B. vietnamiensis G4, an opportunistic pathogen as the model organism. Both gel-based (LC-MS/MS) and gel-free MudPIT (LC/LC-MS/MS) approaches have been applied for the analysis of the proteins extracted from four different cellular fractions of these bacteria. More than 1200 proteins were identified from these analyses, including many proteins previously identified as virulence factors of these bacteria. Similar methodologies were applied to build a proteome map of non-pathogenic B. thailandensis E264 to use as a reference for the pathogenic studies. Additionally, proteomes of two B. thailandensis strains isolated from two geographical locations were compared to investigate the differences in protein expression of these organisms.Proteins identified from pathogenic B. pseudomallei were compared with the non-pathogenic B. thailandensis and opportunistic pathogen B. vietnamiensis proteins. Many species specific proteins were identified from this proteomic analyses; those proteins can be used as antigen targets to selectively identify these pathogenic bacteria in a complex biological matrix using affinity capture methods.Ticks are vectors that can transmit disease causing pathogens one host to another. Knowing the pathogen reservoir is important in order to control disease spread in the environment. Application of mass spectrometric methods to identify the host blood components from tick vectors was investigated using tick nymphs which feed only once in their life cycle. Using mass spectrometry based proteomics; host specific proteins like hemoglobin and immunoglobulin were identified from a single tick nymph analysis. Additional studies have examined the fatty acid profiles of rabbit and sheep blood fed tick nymphs using SPALDI mass spectrometry. Different fatty acid profiles were obtained for these tick nymphs, but further investigations are required to validate these findings
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Identification of Residual Blood Proteins in Ticks by Mass Spectrometry Proteomics
Mass spectrometry–based proteomics of individual ticks demonstrated persistence of mammalian host blood components, including α- and β-globin chains, histones, and mitochondrial enzymes, in Ixodes scapularis and Amblyomma americanum ticks for months after molting. Residual host proteins may identify sources of infection for ticks
One-Hour Screening of Adulterated Heparin by Simplified Peroxide Digestion and Fast RPIP-LC-MS<sup>2</sup>
Early
detection of potential contaminants in heparin, an extensively
used anticoagulant in drug formulations and medical devices, is critical
to ensuring public health. In response to heparin adulteration by
oversulfated chondroitin sulfates (OSCS) that was associated with
adverse events including deaths in 2007–2008, many methods
have been developed to detect OSCS in heparin. However, an analytical
challenge for quality screenings has been to speed up these measurements
to address the complex distribution scheme of heparin in today’s
global market. Here an approach based on mass spectrometry is described
that enables the measurement of adulterated heparin in 1 h, significantly
shortening the time frame of screening for potential contaminants.
The methodology is based on simplified peroxide digestion that rapidly
depolymerizes large polysaccharide chains to small oligosaccharides
followed by fast liquid chromatography mass spectrometry to determine
sample purity. We find that rapid peroxide digestion generates abundant
C- and Y-type oligosaccharides that can be used to differentiate parent
glycosaminoglycans via unsupervised multivariate analysis, including
heparin, chondroitin sulfate A, dermatan sulfate, and the infamous
OSCS. With quantitation demonstrated at 1% (w/w), or 50 ng, OSCS in
heparin and the lower limit of detection estimated at ∼0.20%
(w/w), or ∼10 ng, OSCS in heparin, the technology was sufficiently
sensitive to differentiate real-life, “authentic” adulterated
heparin samples and to quantify this contaminant with an error <10%
relative standard deviation. The methodologies presented here are
deliberately simple to foster adoption and increase the analytical
throughput of mass spectrometric screening in the routine quality
assessment of heparin and other types of compounds of this molecular
family
Assessment of global proteome in LNCaP cells by 2D-RP/RP LC–MS/MS following sulforaphane exposure
The phytochemical sulforaphane can induce cell cycle arrest and apoptosis in metastatic prostate cancer cells, though the mechanism of action is not fully known. We conducted a global proteome analysis in LNCaP metastatic prostate cancer cells to characterize how global protein signature responds to sulforaphane. We conducted parallel analyses to evaluate semi-quantitative 1-dimensional versus 2-dimensional liquid chromatography tandem mass spectrometry (LC–MS/MS) and their utility in characterizing whole cell lysate. We show that 2-dimensional LC–MS/MS can be a useful tool for characterizing global protein profiles and identify TRIAP1 as a novel regulator of cell proliferation in LNCaP metastatic prostate cancer cells. Keywords: Prostate cancer, Sulforaphane, Mass spectrometr
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BrachaShayVetMedMultiplexBiomarkerApproach.pdf
Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. Commercial screening tests lack specificity and current diagnostic procedures are invasive. A proof of concept pilot project for analyzing the canine urinary proteome as a non-invasive diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of TCC, predicted the presence of disease with 90% accuracy.Keywords: Canine, Biomarkers, Tandem mass spectrometry, Liquid chromatography, Transitional cell carcinomaKeywords: Canine, Biomarkers, Tandem mass spectrometry, Liquid chromatography, Transitional cell carcinom