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:10993 5 page(s) 2010-12-21T18:40:08.314Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:10970 A robust method has been developed that allows analysis of both N- and O-linked oligosaccharides released from glycoproteins separated using 2D-PAGE and then electroblotted to PVDF membrane. This analysis provides efficient oligosaccharide profiling applicable to glycoproteomic analysis. The method involves the enzymatic release of N-linked oligosaccharides using PNGase F followed by the chemical release of O-linked oligosaccharides using reductive β-elimination and analysis using LC−ESI−MS. Oligosaccharides from the major plasma glycoproteins with a pI between 4 and 7 were characterized from the glycoforms of haptoglobin, α₂-HS-glycoprotein, serotransferrin, α₁-antitrypsin, and α₁-antichymotrypsin. It was shown that the separation of protein glycoforms evident in 2D-PAGE is partially due to the combined sialylation of the O-linked and N-linked oligosaccharides. Bi-, tri- and tetra-antennary N-linked structures, which had differing levels of sialylation and fucosylation, were found to be present on the glycoproteins analyzed, together with O-linked oligosaccharides such as mono-, and disialylated T-antigen and a disialylated core type 2 hexasaccharide. In addition, N-linked site-specific information was obtained by MALDI-MS analysis using tryptic digestion after PNGase F release of the oligosaccharides. 2010-12-16T08:10:35.291Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:10964 10 page(s) 2010-12-15T16:21:14.578Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:10961 Reducing O-linked oligosaccharides from bovine submaxillary mucin, bovine fetuin, and porcine gastric mucin were recovered by nonreductive alkaline β-elimination from an in-line flow system. Glycoproteins where attached to a solid support using hydrophobic interaction with alkali-resistant Poros reversed phase beads and a flow of alkali released the oligosaccharides. The alkali was subsequently neutralized by a continuous flow through cation exchange resin. The released oligosaccharides in the flow were trapped in a cartridge filled with graphitized carbon. Salt-free oligosaccharides could be recovered as a concentrated solution by elution with organic solvents from the cartridge. The glycosylation pattern of the released oligosaccharides was compared with the conventionally released and reduced oligosaccharides recovered from alkaline β-elimination in the presence of borohydride. In general, the recovery from the in-line release was sometimes lower than from the reductive elimination method, but it was shown that alkaline degradation of reducing oligosaccharides was limited in this system. Liquid chromatography using graphitized carbon packing and high pH mobile phases together with negative ion electrospray mass spectrometry showed that both neutral and acidic reducing oligosaccharides could be analyzed in a single run. Reducing O-linked oligosaccharides could also be recovered in this way from human glycophorin separated by SDS–PAGE. The polyacrylamide was sufficient to retain the glycoprotein in the gel while the flow of alkali released the oligosaccharides. It was also shown that the alkaline conditions for releasing O-linked oligosaccharides from fetuin would partially release some N-linked oligosaccharides, particularly in the presence of reducing agent. 2010-12-15T16:20:32.129Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:10962 A technique with subpicomolar sensitivity was developed for analyzing O-linked oligosaccharides released from glycoproteins separated by gel electrophoresis. The protocol involves gel electrophoresis, electroblotting to poly(vinylidene fluoride) membrane, reductive β-elimination, and analysis of released oligosaccharides by liquid chromatography coupled to negative ion electrospray mass spectrometry. It was also found that N-linked oligosaccharides could be recovered under the same conditions, found both as free oligosaccharides and as distinct glycopeptides created from reductive cleavage of the protein backbone, giving some information on site-specific glycosylation. The method was used to demonstrate that the difference between human α-2HS-glycoprotein isoforms separated by 2D-gel electrophoresis was partially due to sialylation of both O-linked and N-linked oligosaccharides. It was also shown that both acidic and neutral oligosaccharides could be recovered and analyzed simultaneously from high molecular mass (200000−5000000 Da) highly glycosylated mucin glycoproteins collected from small intestine and saliva and separated by sodium dodecyl sulfate−agarose/polyacrylamide composite gels. Mass spectrometric data not only gave information about the mass distribution of the heterogeneous mixtures of oligosaccharides from [M − xH]x- ions but also gave information about the isomeric heterogeneity of the oligosaccharides from their resolution by porous graphitized carbon chromatography. Tandem mass spectrometry was explored as a technique for distinguishing between oligosaccharide isomers with different sequences and also between oligosaccharides with the same sequence but with different linkage configurations. 2010-12-15T16:20:26.544Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:5968 Myosin binding protein C (MyBPC) is a sarcomeric protein whose role in sarcomere structure and regulation of contraction is currently under investigation. It is a member of the immunoglobulin superfamily and is found in the C-zone of the A-band of the sarcomere. The elongated structure of MyBPC is composed of a series of immunoglobulin and fibronectin domains, with the C-terminal domains binding to the myosin thick filament and the N-terminal domains interacting with the myosin subfragment-2 (S2) neck region and possibly the actin thin filament. The functions of MyBPC are to stabilise the sarcomere structure and to regulate contraction. When phosphorylated near its N-terminus, MyBPC no longer binds myosin-S2, causing an increase in the ordering of the myosin heads, ATPase activity, Fmax and Ca²⁺ sensitivity of contraction. Mutations in MyBPC have been found to cause familial hypertrophic cardiomyopathy (FHC) and changes in MyBPC phosphorylation have been linked to cardiac ischaemia-reperfusion injury. 2010-01-27T22:24:00.706Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:5977 In this review we examine the current state of analytical methods used for shotgun proteomics experiments in plants. The rapid advances in this field in recent years are discussed, and contrasted with experiments performed using current widely used procedures. We also examine the use of subcellular fractionation approaches as they apply to plant proteomics, and discuss how appropriate sample preparation can produce a great increase in proteome coverage in subsequent analysis. We conclude that the conjunction of these two techniques represents a significant advance in plant proteomics, and the future of plant biology research will continue to be enriched by the ongoing development of proteomic analytical technology 2010-01-27T22:23:53.172Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:5986 Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs. 2010-01-27T22:23:40.898Z ]]> http://www.researchonline.mq.edu.au/vital/access/manager/Repository/mq:5988 A novel in-gel endoglycosidase technique to study oligosaccharides with graphitized carbon LC−MS has revealed differences in the sulfation profile between the linkage and repeat regions of chondroitin sulfate on aggrecan. Bovine articular cartilage aggrecan was isolated in a composite agarose PAGE gel or diluted in ammonium acetate buffer and was digested overnight with chondroitinase ABC. Including a chemical release/reduction protocol after digestion, we could separate and detect three differentially sulfated chondroitin sulfate disaccharides of the repeat region (ΔUA1-3GalNAc0/4/6S-ol) from the three differentially sulfated linkage region hexasaccharides (ΔUA1-3GalNAc0/4/6Sβ1-4GlcAβ1-3Galβ1-3Galβ1-4Xylitol). Graphitized carbon LC−MS in the negative ion mode was able to resolve isomeric disaccharides and linkage region hexasaccharides. Specific MS² and MS³ enabled us to confirm the sulfate location on all oligosaccharides by comparing their fragmentation with sulfated disaccharide standards. The presence of unsulfated, 6-sulfated, and 4-sulfated linkage regions was correlated with positive Western blot staining with the respective CS linkage region neoepitope antibodies (1B5, 3B3, 2B6) on digested aggrecan. Our strategy of examining linkage region and repeat region profiles is applicable to screening GAGs from various biological samples in order to detect differences between normal and disease states. 2010-01-27T22:23:39.664Z ]]>