Commentary on Section 264 of ITAA 1936: commissioner may require information and evidence
Dabner, Justin
2007-01-01
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62 records were found.
Accurate quantitation of analytes in simple or complex matrices is imperative for detailed understandings of biological or synthetic systems and is also necessary to ensure consumer safety with regards to food, pharmaceutical formulations, and environmental hazards. Liquid chromatography-mass spectrometry (LC-MS) is capable of completing such tasks using a variety of quantitative methods. In Chapter 1, these methods are presented with regards to chemical warfare agent studies. External calibration, arguably the simplest of these techniques, quantifies an analyte by comparing the analyte's response in the sample matrix to the analyte's response in standard solutions. The success of this method hinges upon the consistency of the analyte's response between the blank and matrix samples. The work in Chapters 2 and 3 is an investigation into...
Glycosylation on proteins serve many biological roles and their heterogeneity can affect various biological functions. The site occupancy of the glycans as well as their various glycoforms makes glycans unique to analyze due to the fact that the variation in structure is not based on a genomic code. Glycosylation is a post-translational modification that provides diverse glycoforms. Mass spectrometry has become a standard technique for characterizing the site occupancy and glycoforms that a protein exhibits. Methodologies in characterization of glycopeptides often include a proteolytic digest followed by purification of the digested protein with liquid chromatography. Mass spectrometers are sensitive to salts and buffer contents, thus a purification of the sample before analysis is vital for the detection of the sample. The research pr...
The mass defect of a substance can be used in mass spectral analysis to identify peaks as likely belonging to a compound class, such as peptides, if the mass defect is within the known range for that compound class. For peptides, a range of possible mass defects was calculated previously, using a set of theoretical peptides, where all possible amino acid combinations were considered (Mann, M. Abstract from the 43rd Annual Conference on Mass Spectrometry and Allied Topics; 1995, ASMS). We compare that range of theoretical peptide mass defects to new values obtained from in silico tryptic digests of proteins that are abundant in human serum and human seminal fluid. The range of mass defect values encompassing 95% of peptides for the human protein data sets was found to be up to 50% smaller than the previously reported mass defect range f...
Protein post-translational modifications (PTMs) are important for a variety of reasons. PTMs confer the final protein product and biological functionality onto a nascent protein chain. Two common PTMs are glycosylation and disulfide bond formation. Both glycosylation and disulfide bond formation contribute to a variety of biological processes, including protein folding and stabilization. Mass spectrometry (MS) has shown to be an essential technique to study PTMs, especially when tandem mass spectrometry (MS/MS) experiments are performed. In the characterization of PTMs using MS/MS, different fragmentation techniques are often used. Regardless of the dissociation method that is employed, MS/MS data interpretation is a tedious and lengthy process. To render this analysis more efficient, the use of automated tools is necessary. In this wo...
Increasing interest in production of protein-based pharmaceuticals (biotherapeutics) is accompanied by an increased need for verification of protein folding and correct disulfide bonding. Recombinant protein expression may produce aberrant disulfide bonds and could result in safety concerns or decreased efficacy. Thus, the thorough analysis of disulfide bonding is a necessity for protein therapeutics. The use of ETD facilitates this analysis because disulfide bonds are preferentially cleaved when subjected to ETD. Here, we make use of this well-characterized reaction to assign disulfide bonding networks by coupling the use of extracted ion chromatograms (XICs) of cysteine-containing peptides with ETD analysis to produce an efficient assignment approach for disulfide bonding. This method can be used to assign a disulfide pattern in a de...
The purpose of this review is to provide those interested in glycosylation analysis with the most updated information on the availability of automated tools for MS characterization of N-linked and O-linked glycosylation types. Specifically, this review describes software tools that facilitate elucidation of glycosylation from MS data on the basis of mass alone, as well as software designed to speed the interpretation of glycan and glycopeptide fragmentation from MS/MS data. This review focuses equally on software designed to interpret the composition of released glycans and on tools to characterize N-linked and O-linked glycopeptides. Several websites have been compiled and described that will be helpful to the reader who is interested in further exploring the described tools.
N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs....
Bcl-2 regulates apoptosis by controlling luminal Ca2+ concentration of endoplasmic reticulum (ER). Dremina et al. reported that Bcl-2 interacts with SERCA, the Ca2+ pump in SR/ER membrane, causing inactivation and translocation. This work reports the characteristics of the SERCA/Bcl-2 interactions using wild type and three mutants, G145E, S24C/C158S and S205C/C158S, of the truncated protein, Bcl-2Δ21. Protein cross-linking, Ca2+-ATPase activity assay, Sucrose Density Gradient fractionation, immunoprecipitation and the fusion protein binding assay are the approaches used. Results reveal that the two proteins can interact with both 1:1 and 2:1 (Bcl-2Δ21: SERCA) molar ratios. The hydrophobic groove of Bcl-2Δ21 is involved in the interactions. The BH1 domain of Bcl-2Δ21 interacts with the ATP binding domain of SERCA. The G145E mutant is a ...
This research offers an alternative approach to the study of tissue-specific metabolism by metabonomics, called tissue-targeted metabonomics. Microdialysis sampling was used to sample small endogenous metabolites in tissue extracellular fluid. Samples were then analyzed by proton nuclear magnetic resonance spectroscopy. The research explored analytical and biological considerations for this method. Data treatment, including integration of the NMR spectra and normalization to standards, was optimized for different dialysate types. Time trends in the basal metabolism of liver and heart tissues were described, as were the metabolic effects of anesthesia, circadian rhythms and animal activity. After the method was developed and basal metabolism was adequately characterized, tissue-targeted metabonomics was used to study oxidative stress, i...
A diverse set of systems that interact with nitric oxide (NO) is important because of its adverse role in environmental chemistry and importance in mammalian biology. Towards this effort, nanomaterials that interact with NO have been developed because they can display beneficial and unique chemical and physical properties from their larger counterparts. While several nanomaterials have been developed that store and release NO, few systems composed entirely of metal complexes have been reported. The research described in this dissertation involves the development of nanomaterials that interact with NO. Molecule-based nanoparticulate metal complexes were prepared using precipitation with compressed antisolvent technology. Microscopy and powder x-ray diffraction were used to determine that the nanoparticles form lamellar structures during...
