http://www.researchonline.mq.edu.au/vital/access/services/Feed ${session.getAttribute("locale")} 5 http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:25634 This publication describes the development of an automated platform for the study of the plasma glycoproteome. The method consists of targeted depletion in-line with glycoprotein fractionation. A key element of this platform is the enabling of high throughput sample processing in a manner that minimizes analytical bias in a clinical sample set. The system, named High Performance Multi-Lectin Affinity Chromatography (HP-MLAC), is composed of a serial configuration of depletion columns containing anti-albumin antibody and protein A with in-line multilectin affinity chromatography (M-LAC) which consists of three mixtures of lectins concanavalin A (ConA), jacalin (JAC), and wheat germ agglutinin (WGA). We have demonstrated that this platform gives high recoveries for the fractionation of the plasma proteome (≥ 95%) and excellent stability (over 200 runs). In addition, glycoproteomes isolated using the HP-MLAC platform were shown to be highly reproducible and glycan specific as demonstrated by rechromatography of selected fractions and proteomic analysis of the unbound (glycoproteome 1) and bound (glycoproteome 2) fractions. 2013-05-20T04:40:19.714Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:25635 TNK-tPA products from the innovator and follow-on manufacturers were characterized and compared. All tryptic peptides including N-terminal, C-terminal, and mutated peptides as well as the disulfide-linked peptides were identified, with the demonstration of the same primary sequence and disulfide linkages between the innovator and follow-on products. The three N-linked and one O-linked fucose glycosylation sites were identified. The two N-linked fucose sites (N103 and N448) and one O-linked fucose site (T61) were fully glycosylated in both innovator and follow-on products. The other N-linked site (N184) was partially glycosylated and exhibited a 2.5-fold difference between the innovator (60% occupancy) and follow-on (25% occupancy) products. Since the glycosylation occupancy at this site is known to affect biological activity in the clot lysis assay, this observed difference could cause a concern as to their bioequivalence. The cleavage site for the conversion of the zymogen form to active enzyme was also identified between R275 and I276, with a cleavage of 40% for the innovator and 10% for the follow-on products. Both the glycosylation occupancy (%) and the chain cleavage (%) were determined by two independent approaches, starting from either the peptide or intact protein separation, with consistent results by both methods. Subtle differences of modifications such as deamidation and oxidation between the innovator and biosimilar products were shown at M207, M445, M490 and N58, N184. The observation of different extents of oxidation at M207 and deamidation at N184, which could influence the clot lysis activity, were also of potential concern in drug efficacy between the follow-on and innovator products. 2013-05-20T04:40:11.005Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:25511 Liquid chromatography-selected reaction monitoring (LC-SRM) is a highly specific and sensitive mass spectrometry (MS) technique that is widely being applied to selectively qualify and validate candidate markers within complex biological samples. However, in order for LC-SRM methods to take on these attributes, target-specific optimization of sample processing is required, in order to reduce analyte complexity, prior to LC-SRM. In this study, we have developed a targeted platform consisting of protein immunoaffinity enrichment on magnetic beads and LC-SRM for measuring carbonic anhydrase 12 (CA12) protein in a renal cell carcinoma (RCC) cell line (PRC3), a candidate biomarker for RCC whose expression at the protein level has not been previously reported. Sample processing and LC-SRM assay were optimized for signature peptides selected as surrogate markers of CA12 protein. Using LC-SRM coupled with stable isotope dilution, we achieved limits of quantitation in the low fmol range sufficient for measuring clinically relevant biomarkers with good intra- and interassay accuracy and precision (≤17%). Our results show that using a quantitative immunoaffinity capture approach provides specific, accurate, and robust assays amenable to high-throughput verification of potential biomarkers. 2013-05-15T14:22:46.406Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:25512 Recombinant tissue plasminogen (rt-PA) with 35 cysteine residues has been completely assigned by mapping the 17 disulfide linkages and the unpaired cysteine. The result is consistent with the prediction from homology except for the unassigned cysteine, which was identified at Cys83. This cysteine was found to be blocked and paired with either a glutathione or cysteine residue in an 60:40 ratio, respectively. The analysis was conducted using a multifragmentation approach consisting of electron transfer dissociation (ETD) and collision induced dissociation (CID), in combination with a multienzyme digestion strategy (Lys-C, trypsin, and Glu-C). The disulfide-linked peptides, even those containing N- or O-linked glycosylation, could be assigned since the disulfide bonds were still preferably cleaved over the glycosidic cleavages under ETD fragmentation. The use of a multiple and sequential enzymatic digestion strategy was important in producing fragment sizes suitable for analysis. For the analysis of complex intertwined disulfides, the use of CID−MS3 to target partially disulfide-dissociated peptides from the ETD fragmentation was necessary for linkage assignment. The ability to identify the exact location and status of the unpaired cysteine (free or blocked with a glutathione or cysteine) could shed light on the activation of rt-PA, upon stimulation by either oxidative or ischemic stress. 2013-05-15T14:22:45.794Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:24249 24 page(s) 2013-02-18T06:31:09.615Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:21312 Protein glycosylation represents one of the major post-translational modifications and can have significant effects on protein function. Moreover, changes in the carbohydrate structure are increasingly being recognized as an important modification associated with cancer etiology. In this report, we describe the development of a proteomics approach to identify breastcancer related changes in either concentration and/or the carbohydrate structures of glycoprotein(s) present in blood samples. Diseased and healthy serum samples were processed by an optimized sample preparation protocol using multiple lectin affinity chromatography (M-LAC) that partitions serum proteins based on glycan characteristics. Subsequently, three separate procedures, 1D SDS-PAGE, isoelectric focusing and an antibody microarray, were applied to identify potential candidate markers for future study. The combination of these three platforms is illustrated in this report with the analysis of control and cancer glycoproteomic fractions. Firstly, a molecular weight based separation of glycoproteins by 1D SDS-PAGE was performed, followed by protein, glycoprotein staining, lectin blotting and LC–MS analysis. To refine or confirm the list of interesting glycoproteins, isoelectric focusing (targeting sialic acid changes) and an antibody microarray (used to detect neutral glycan shifts) were selected as the orthogonal methods. As a result, several glycoproteins including alpha-1B-glycoprotein, complement C3, alpha-1-antitrypsin and transferrin were identified as potential candidates for further study. 2012-09-12T18:32:59.682Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:16046 A nanotube electronic needle biosensor was developed to provide fast, low cost, accurate detection of biomolecules. The sensor was formed by synthesizing highly aligned multi-wall carbon nanotube arrays. Nanotube bundles from the array were welded onto the tips of tungsten needles using a microscope. The needles were then encased in glass and a polymer coating. Cyclic voltammetry (CV) for the respective reduction of 6 mM K3Fe(CN)6in a 1.0 M KNO3was performed to examine the redox behavior of the nanotube needle. The CV results showed a steady-state response attributable to radial diffusion with a high steady-state current density. An amperometric sensor was then developed for glucose detection by physical attachment of glucose oxidase on the nanotube needle. A label-free immunosensor based on electrochemical impedance spectroscopy was also formed. The nanotube needle amperometric have good sensitivity with a low detection limit, and the possibility exists to keep decreasing the size of the needle to increase the sensitivity. 2011-11-21T23:10:38.112Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:14197 A Ni(II)tetrakis(4-sulfonatophenyl) porphyrin (NiTPPS)|carbon nanotube composite electrode that shows strong catalytic and antifouling capability was developed to detect a series of phenolic endocrine compounds including bisphenol A, nonylphenol and ethynylestradiol. This electrode was fabricated by electropolymerizing NiTPPS complexes on a carbon nanotube-modified glassy carbon electrode. Optimized experimental parameters including a hydrodynamic potential of 0.7 V for flow injection analysis (FIA) and a NiTPPS surface coverage of 2.2 nmol cm⁻² (standard deviation 0.2 nmol cm⁻²; n = 6) were obtained for detection of the endocrine disrupting compounds. The sensor responded well to all the tested compounds with limits of detection ranging from 15 nmol L⁻¹ to 260 nmol L⁻¹ (based on three times S/N ratio) under FIA conditions. Both carbon nanotubes and NiTPPS account for the excellent performance of the composite modified electrode. 2011-08-01T06:25:31.785Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:13422 8 page(s) 2011-05-30T14:10:21.368Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:13417 Because highly luminescent lanthanide compounds are limited to Eu(3+) and Tb(3+) compounds with red (Eu, approximately 615 nm) and green (Tb, approximately 545 nm) emission colors, the development and application of time-resolved luminescence bioassay technique using lanthanide-based multicolor luminescent biolabels have rarely been investigated. In this work, a series of lanthanide complexes covalently bound silica nanoparticles with an excitation maximum wavelength at 335 nm and red, orange, yellow and green emission colors has been prepared by co-binding different molar ratios of luminescent Eu(3+)-Tb(3+) complexes with a ligand N,N,N(1),N(1)-(4'-phenyl-2,2':6',2''-terpyridine-6,6''-diyl)bis(methylenenitrilo) tetrakis (acetic acid) inside the silica nanoparticles. The nanoparticles characterized by transmission electron microscopy and luminescence spectroscopy methods were used for streptavidin labeling, and time-resolved fluoroimmunoassay (TR-FIA) of human prostate-specific antigen (PSA) as well as time-resolved luminescence imaging detection of an environmental pathogen, Giardia lamblia. The results demonstrated the utility of the new multicolor luminescent lanthanide nanoparticles for time-resolved luminescence bioassays. 2011-05-30T02:50:05.730Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:13411 We describe an optical chemical sensor using a multi-channel directional coupler with slot waveguides. 2011-05-27T17:50:18.325Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:13160 Full network level privacy has often been categorized into four sub-categories: Identity, Route, Location and Data privacy. Achieving full network level privacy is a critical and challenging problem due to the constraints imposed by the sensor nodes (e.g., energy, memory and computation power), sensor networks (e.g., mobility and topology) and QoS issues (e.g., packet reach-ability and timeliness). In this paper, we proposed two new identity, route and location privacy algorithms and data privacy mechanism that addresses this problem. The proposed solutions provide additional trustworthiness and reliability at modest cost of memory and energy. Also, we proved that our proposed solutions provide protection against various privacy disclosure attacks, such as eavesdropping and hop-by-hop trace back attacks. 2011-05-25T21:45:39.919Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:12602 Glycopeptide enrichment is a prerequisite to enable structural characterization of protein glycosylation in glycoproteomics. Here we present an improved method for glycopeptide enrichment based on zwitter-ionic hydrophilic interaction chromatography solid phase extraction (ZIC-HILIC SPE) in a microcolumn format. The method involves TFA ion pairing (IP) to increase the hydrophilicity difference between glycopeptides and nonglycosylated peptides. Three mobile phases were investigated, i.e., 2% formic acid (defined as IP2% FA ZIC-HILIC SPE), 0.1% TFA and 1% TFA (defined as IP0.1% TFA and IP1% TFA ZIC-HILIC SPE) all containing 80% acetonitrile. Samples of increasing complexities, i.e., digests of single glycoproteins, a five-glycoprotein mixture, and depleted plasma, were used in the study. The presence of TFA in the mobile phase significantly improved the glycopeptide enrichment for all complexities, as evaluated by enhanced glycopeptide detection using MALDI-TOF MS and RP-LC−ESI-MS/MS, e.g., the glycopeptide ion signals were increased by up to 3.7-fold compared to IP2% FA conditions. The enhanced glycopeptide detection was promoted by a substantial depletion of nonglycosylated peptides, offering an almost complete isolation of IgG glycopeptides using a single SPE enrichment step and a reduction from 711 nonglycosylated peptides observed in the IP2% FA ZIC-HILIC SPE retained plasma fraction, to only 157 and 97 when 0.1% and 1% TFA was used in the mobile phase. In conclusion, this systematic study has shown that TFA-containing mobile phases increase glycopeptide enrichment efficiency considerably for a broad range of sample complexities when using ZIC-HILIC SPE. 2011-04-13T05:30:30.427Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:12105 We investigate the use of multi-channel directional couplers as evanescent field optical sensors. If the surrounding analyte can infiltrate the region between the array waveguides then these sensors can exploit the exponential dependence of coupling on the analyte refractive index. Analysis based on supermodes and the principle of self-imaging is used to develop design guidelines for optimizing sensor performance. It is shown that the quasi-periodic characteristic of these sensors facilitates a trade-off between sensitivity and device length. 2011-03-07T06:10:15.624Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:11888 Urinary tract infections (UTIs) are known to alter the normal urine composition which, in principle, can lead to changes in urine autofluorescence. This paper describes the study of human urine (normal and UTI) by using UV fluorescence excitation/emission matrices and synchronous spectra and proposes a method of diagnosing UTI without any sample preparation. The method is based on excitation in the shorter UV region (250–350 nm) which shows good discrimination between the normal urine and UTI samples. The synchronous scans with an offset of Δλ = 90 nm were also able to differentiate between normal urines and UTI samples. These differences were observed even though the two known major urine fluorophores, tryptophan and indoxyl sulfate were present in the normal urine and UTI samples in similar concentration as established by HPLC analysis. Although the identity of substances responsible for the altered autofluorescence in UTI is not established, our study shows that autofluorescence has the potential to differentiate between normal human urine samples and those with UTI. 2011-02-21T21:10:38.569Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:11756 Despite the maturation of organic chemistry and the efforts of medicinal and combinatorial chemistry, natural products play a preeminent role in drug discovery. However, there is a general lack of information concerning cellular targets of most natural products but, without an understanding of how biologically active natural products work, efforts to generate more potent analogs through structure–activity relationships, structure-based design, or combinatorial chemistry is made more difficult. Similarly important, but even less well known, are the identities of ‘off-targets’ that are responsible for toxicity or side effects of natural product drugs. Chemical proteomics approaches, especially affinity chromatography, dominate the methods used to identify natural product-binding proteins. However, reverse chemical proteomics methods such as phage, mRNA, and ribosome displays have begun to emerge and others such as cell display, protein and chemical arrays, yeast three-hybrid, the viral and DNA displays are methods that hold the promise of being able to rapidly identify natural product-binding proteins. In this chapter, we review various methods that have been used to determine the binding proteins for natural products but also look critically at methods that might be used in the near future to achieve this goal. Each has advantages and disadvantages and there is no one perfect method, but many are complementary. This is certainly a rapidly evolving and exciting facet of natural products research. 2011-02-10T09:40:31.817Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:11363 14 page(s) 2011-01-19T04:30:35.723Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:10981 Negative ion nano-liquid chromatography/mass spectrometry (nano-LC/MS) and tandem mass spectrometry (nano-LC/MS2), using graphitised carbon as separating medium, were explored for analysing neutral and acidic O-linked and N-linked oligosaccharide alditols. Compared to the sensitivity of capillary LC/MS (flow rate of 6 μL/min) coupled with a conventional electrospray ionisation source, the nano-LC/MS (flow rate of 0.6 μL/min) with a nanoflow ion source was shown to increase the sensitivity ten-fold with a detection limit in the low-femtomole range. The absolute signals for the [MnH]n− ions of the oligosaccharides were increased 100-fold, enabling accumulation of high-quality fragmentation data in MS2 mode, in which detection of low abundant sequence ions is necessary for characterisation of highly sialylated N-linked oligosaccharides. Oligosaccharides with high numbers of sialic acid residues gave dominant fragments arising from the loss of sialic acid, and less abundant fragments from cleavage of other glycosidic bonds. Enzymatic off-line desialylation of oligosaccharides in the low-femtomole range prior to MS2 analysis was shown to increase the quality of the spectra. Automated glycofragment mass fingerprinting using the GlycosidIQ software confirmed the oligosaccharide sequence for both neutral desialylated as well as sialylated structures. Furthermore, the use of graphitised carbon nano-LC/MS enabled the detection of four sialylated O-linked oligosaccharides on membrane proteins from ovarian tissue (5 μg of total amount of protein). 2010-12-17T01:20:19.365Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:10975 Capillary liquid chromatography–mass spectrometry using graphitised carbon stationary phase and ion trap mass spectrometry was shown to be a powerful technique for analysing glycosaminoglycans digested with endoglycosidases. Commonly found disaccharides from heparin/heparan sulphate digests at sub nanomole levels were found to be separated by mass and/or retention time and detected by negative ion electrospray mass spectrometry predominantly as [M − H]− ions using a standard electrospray interface and flow rate between 6–10 μL/min. Graphitised carbon liquid chromatography–fragmentation mass spectrometry provided sequence data of disaccharides and oligosaccharides. Sequence information was obtained from either collision of the [M − H]− ions (low sulphated disaccharides) or of the [M + Na − 2H]− ions (highly sulphated disaccharides). This separation and identification method of endoglycosidase digestion and sample preparation using a combination of cation exchange and graphitised carbon, was used to successfully analyse digests of keratan sulphate (keratanase) and heparin (heparinase) standards, and hyaluronic acid (hyaluronidase) from synovial fluid samples. 2010-12-16T08:10:19.210Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:9302 Templated electrodeposition of gold to produce thin (<1 μm) films containing a close packed hexagonal array of uniform sphere segment voids is shown to give surfaces which show strong surface enhancement for Raman scattering from molecules adsorbed at the surface. The magnitude of this is enhancement is determined by the precise geometry of the surface and depends on the choice of void diameter and film thickness. The resulting SER active surfaces are stable, reusable, give reproducible surface enhancement and can be used for in situ electrochemical SERS studies. 2010-08-29T12:10:10.103Z ]]>