34 research outputs found
Studies on natural product derivatives : HIV therapies incorporating marine natural products.
No full textCV-N is an 11 kDa, anti-HIV protein that binds strongly to the envelope glycoprotein, gp120, expressed on the outer surface of the free virion and also on HIV-infected cells. As such, it represents an important lead for development of anti-HIV therapeutics. Marine toxins such as the halichondrins have potent in vivo cytotoxicities and are lethal to cells. The combination of this potency of the marine toxins with the unique targeting capability of CV-N has been harnessed to produce conjugates that have the potential to selectively target and eliminate HIV-infected cells.
Three forms of the protein were developed; the native protein itself, a derivative recombinantly produced in E. coli with an extra cysteine at the C-terminal (CV-N-Cys) and CV-N with the lysine side chain amines converted into thiols (thiolated-CV-N).
To facilitate release of the toxin within infected cells an enzymatically-cleavable pHdependent biolinker was incorporated separating the toxin from the protein. The chemistry required for incorporation of protein, biolinker, and toxin, was established through synthesis of fluorescently labelled conjugates capable of reaction with CV-N. Biological testing of these derivatives showed no interference with the anti-HIV activity of the CV-N when conjugated in these model compounds. Synthetic strategies were developed to produce two derivatives of norhomohalichondrin B amine, both containing the cleavable biolinker, but with activation from succinimidyl esters and maleimido groups respectively.
Native CV-N was reacted with the succinimidyl ester derived toxin construct to produce a CV-N-biolinker-toxin conjugate. The maleimido derivative toxin construct was reacted with both CV-N-Cys and thiolated-CV -N to produce closely related CV-N-toxin conjugates. Investigations into the binding properties and cell toxicities of these conjugates is currently underway
The New and the Old: Platform Cross-Validation of Immunoaffinity MASS Spectrometry versus ELISA for PromarkerD, a Predictive Test for Diabetic Kidney Disease
No full textPromarkerD is a proteomics derived test for predicting diabetic kidney disease that measures the concentrations of three plasma protein biomarkers, APOA4, CD5L and IBP3. Antibodies against these proteins were developed and applied to a multiplexed immunoaffinity capture mass spectrometry assay. In parallel, and facilitating current clinical laboratory workflows, a standard ELISA was also developed to measure each protein. The performance characteristics of the two technology platforms were compared using a cohort of 100 samples, with PromarkerD test scores demonstrating a high correlation (R = 0.97). These technologies illustrate the potential for large scale, high throughput clinical applications of proteomics now and into the future
Reverse phase HPLC method for detection and quantification of lupin seed ?-conglutin
No full text© 2017 Elsevier B.V. A simple, selective and accurate reverse phase HPLC method was developed for detection and quantitation of ?-conglutin from lupin seed extract. A linear gradient of water and acetonitrile containing trifluoroacetic acid (TFA) on a reverse phase column (Agilent Zorbax 300SB C-18), with a flow rate of 0.8 ml/min was able to produce a sharp and symmetric peak of ?-conglutin with a retention time at 29.16 min. The identity of ?-conglutin in the peak was confirmed by mass spectrometry (MS/MS identification) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The data obtained from MS/MS analysis was matched against the specified database to obtain the exact match for the protein of interest. The proposed method was validated in terms of specificity, linearity, sensitivity, precision, recovery and accuracy. The analytical parameters revealed that the validated method was capable of selectively performing a good chromatographic separation of ?-conglutin from the lupin seed extract with no interference of the matrix. The detection and quantitation limit of ?-conglutin were found to be 2.68 µg/ml and 8.12 µg/ml respectively. The accuracy (precision and recovery) analysis of the method was conducted under repeatable conditions on different days. Intra-day and inter-day precision values less than 0.5% and recovery greater than 97% indicated high precision and accuracy of the method for analysis of ?-conglutin. The method validation findings were reproducible and can be successfully applied for routine analysis of ?-conglutin from lupin seed extract
The Photoyellowing of Stilbene‐derived Fluorescent Whitening Agents—Mass Spectrometric Characterization of Yellow Photoproducts
No full textPromarkerD Versus Standard of Care Biochemical Measures for Assessing Future Renal Function Decline in Type 2 Diabetes
No full textBackground/Objectives: The current standard of care for assessing chronic kidney disease complicating diabetes (DKD) includes measurement of estimated glomerular filtration rate (eGFR) and urinary albumin:creatinine ratio (uACR) but both tests have limitations. The present study compared the biomarker-based Promarker®D test with conventional biochemical measures for predicting future kidney function decline in adults with type 2 diabetes (T2D). Methods: Baseline concentrations of apolipoprotein A-IV, CD5 antigen-like protein and insulin-like growth factor binding protein 3 were combined with age, serum HDL cholesterol and eGFR to generate PromarkerD risk scores for incident DKD/eGFR decline ≥ 30% (the primary endpoint) in 857 adults with T2D (mean age 65.4 years, 54% males). Logistic regression modelling was used to compare the association of (i) PromarkerD, (ii) eGFR, (iii) uACR, and (iv) eGFR plus uACR with this outcome during 4 years of follow-up. Results: Study participants were classified by PromarkerD as low (63%), moderate (13%), or high risk (24%) for kidney function decline at baseline. Over a mean 4.2 years, 12.5% developed the primary endpoint. PromarkerD scores showed significantly higher predictive performance (area under the receiver operating characteristic curve (AUC) 0.88 (95% confidence interval (CI) 0.85–0.91)) compared to conventional biochemical measures (AUC = 0.63–0.82). There was a progressive increase in risk with moderate and high risk by PromarkerD exhibiting greater odds of the primary endpoint compared to those at low risk (odds ratios (OR) (95% CI) 8.11 (3.99–16.94) and 21.34 (12.03–40.54), respectively, both p < 0.001). Conclusions: PromarkerD more accurately identifies adults with T2D at risk of kidney function decline than current usual care biochemical tests
Deep proteogenomics; high throughput gene validation by multidimensional liquid chromatography and mass spectrometry of proteins from the fungal wheat pathogen Stagonospora nodorum
No full textBACKGROUND: Stagonospora nodorum, a fungal ascomycete in the class dothideomycetes, is a
damaging pathogen of wheat. It is a model for necrotrophic fungi that cause necrotic symptoms via
the interaction of multiple effector proteins with cultivar-specific receptors. A draft genome
sequence and annotation was published in 2007. A second-pass gene prediction using a training set
of 795 fully EST-supported genes predicted a total of 10762 version 2 nuclear-encoded genes, with
an additional 5354 less reliable version 1 genes also retained.
RESULTS: In this study, we subjected soluble mycelial proteins to proteolysis followed by 2D LC
MALDI-MS/MS. Comparison of the detected peptides with the gene models validated 2134 genes.
62% of these genes (1324) were not supported by prior EST evidence. Of the 2134 validated genes,
all but 188 were version 2 annotations. Statistical analysis of the validated gene models revealed a
preponderance of cytoplasmic and nuclear localised proteins, and proteins with intracellularassociated
GO terms. These statistical associations are consistent with the source of the peptides
used in the study. Comparison with a 6-frame translation of the S. nodorum genome assembly
confirmed 905 existing gene annotations (including 119 not previously confirmed) and provided
evidence supporting 144 genes with coding exon frameshift modifications, 604 genes with
extensions of coding exons into annotated introns or untranslated regions (UTRs), 3 new gene
annotations which were supported by tblastn to NR, and 44 potential new genes residing within
un-assembled regions of the genome.
CONCLUSION: We conclude that 2D LC MALDI-MS/MS is a powerful, rapid and economical tool to
aid in the annotation of fungal genomic assemblies
Limiting the Hydrolysis and Oxidation of Maleimide–Peptide Adducts Improves Detection of Protein Thiol Oxidation
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