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Djidja M.-C.,Sheffield Hallam University | Claude E.,Waters Corporation | Snel M.F.,Lysosomal Diseases Research Unit | Francese S.,Sheffield Hallam University | And 3 more authors.
Analytical and Bioanalytical Chemistry | Year: 2010

The development of tissue micro-array (TMA) technologies provides insights into high-throughput analysis of proteomics patterns from a large number of archived tumour samples. In the work reported here, matrix-assisted laser desorption/ionisation-ion mobility separation-mass spectrometry (MALDI-IMS-MS) profiling and imaging methodology has been used to visualise the distribution of several peptides and identify them directly from TMA sections after on-tissue tryptic digestion. A novel approach that combines MALDI-IMS-MSI and principal component analysis-discriminant analysis (PCA-DA) is described, which has the aim of generating tumour classification models based on protein profile patterns. The molecular classification models obtained by PCA-DA have been validated by applying the same statistical analysis to other tissue cores and patient samples. The ability to correlate proteomic information obtained from samples with known and/or unknown clinical outcome by statistical analysis is of great importance, since it may lead to a better understanding of tumour progression and aggressiveness and hence improve diagnosis, prognosis as well as therapeutic treatments. The selectivity, robustness and current limitations of the methodology are discussed. © 2010 Springer-Verlag. Source

Fuller M.,Lysosomal Diseases Research Unit | Fuller M.,University of Adelaide
Lipids in Health and Disease | Year: 2010

Sphingolipids constitute a diverse array of lipids in which fatty acids are linked through amide bonds to a long-chain base, and, structurally, they form the building blocks of eukaryotic membranes. Ceramide is the simplest and serves as a precursor for the synthesis of the three main types of complex sphingolipids; sphingomyelins, glycosphingolipids and gangliosides. Sphingolipids are no longer considered mere structural spectators, but bioactive molecules with functions beyond providing a mechanically stable and chemically resistant barrier to a diverse array of cellular processes. Although sphingolipids form a somewhat minor component of the total cellular lipid pool, their accumulation in certain cells forms the basis of many diseases. Human diseases caused by alterations in the metabolism of sphingolipids are conventionally inborn errors of degradation, the most common being Gaucher disease, in which the catabolism of glucosylceramide is defective and accumulates. Insulin resistance has been reported in patients with Gaucher disease and this article presents evidence that this is due to perturbations in the metabolism of sphingolipids. Ceramide and the more complex sphingolipids, the gangliosides, are constituents of specialised membrane microdomains termed lipid rafts. Lipid rafts play a role in facilitating and regulating lipid and protein interactions in cells, and their unique lipid composition enables them to carry out this role. The lipid composition of rafts is altered in cell models of Gaucher disease which may be responsible for impaired lipid and protein sorting observed in this disorder, and consequently pathology. Lipid rafts are also necessary for correct insulin signalling, and a perturbed lipid raft composition may impair insulin signalling. Unravelling common nodes of interaction between insulin resistance and Gaucher disease may lead to a better understanding of the biochemical mechanisms behind pathology. © 2010 Fuller; licensee BioMed Central Ltd. Source

Dawson G.,University of Chicago | Fuller M.,Lysosomal Diseases Research Unit | Helmsley K.M.,Lysosomal Diseases Research Unit | Hopwood J.J.,Lysosomal Diseases Research Unit
Neurochemical Research | Year: 2012

Allogenic stem cell transplantation can reduce lysosomal storage of heparan sulfate-derived oligosaccharides by up to 27 % in Sanfilippo MPS3a brain, but does not reduce the abnormal storage of sialolactosylceramide (GM3) or improve neurological symptoms, suggesting that ganglioside storage is in a non-lysosomal compartment. To investigate this further we isolated the Triton X100-insoluble at 4 °C, lipid raft (LR) fraction from a sucrosedensity gradient from cerebral hemispheres of a 7 month old mouse model of Sanfilippo MPS3a and age-matched control mouse brain. HPLC/MS/MS analysis revealed the expected enrichment of normal complex gangliosides, ceramides, galatosylceramides and sphingomyelin enrichment in this LR fraction. The abnormal HS-derived oligosaccharide storage material was in the Triton X100-soluble at 4 °C fractions (8-12), whereas both GM3 and sialo[GalNAc]lactosylceramide (GM2) were found exclusively in the LR fraction (fractions 3 and 4) and were >90 % C18:0 fatty acid, suggesting a neuronal origin. Further analysis also revealed a >threefold increase in the late-endosome marker bis (monoacylglycerol) phosphate (>70 % as C22:6/22:6-BMP) in non-LR fractions 8-12 whereas different forms of the proposed BMP precursor, phosphatidylglycerol (PG) were in both LR and non-LR fractions and were less elevated in MPS3a brain. Thus heparan sulfate-derived oligosaccharide storage is associated with abnormal lipid accumulation in both lysosomal (BMP) and non-lysosomal (GM3 and GM2) compartments. © Springer Science+Business Media, LLC 2012. Source

Karageorgos L.,Lysosomal Diseases Research Unit | Lancaster M.J.,Australian Department of Primary Industries and Fisheries | Nimmo J.S.,ASAP Laboratory | Hopwood J.J.,Lysosomal Diseases Research Unit
Journal of Inherited Metabolic Disease | Year: 2011

Gaucher disease, an autosomal recessive lysosomal storage disorder caused by mutations in the β-glucocerebrosidase gene, was recently discovered in sheep on a "Southdown" sheep stud in Victoria, Australia. Clinical signs include neuropathy, thickened leathery skin, and ichthyosis, with lambs unable to stand from birth. Affected lambs were found to be deficient in glucocerebrosidase activity, and mutational analysis found them to be homozygous for the missense mutations c.1142G>A (p. C381Y) and c.1400C>T (p.P467L). In addition, four silent mutations were detected (c.777C>A [p.Y259Y], c1203A>G [p.Q401Q], c.1335T>C [p.I445I], c.1464C>G [p.L488L]). The human equivalent [C342Y] to the C381Y mutation leads to an acute neuronopathic phenotype in patients. Identification of an acute neuronopathic form of Gaucher disease in sheep provides a large animal model that will enable studies of pathology and evaluation of therapies to treat this common lysosomal storage disorder. © SSIEM and Springer 2010. Source

Snel M.F.,Lysosomal Diseases Research Unit | Fuller M.,Lysosomal Diseases Research Unit
Analytical Chemistry | Year: 2010

MALDI mass spectrometric imaging (MSI) enables spatially resolved mass and intensity information to be obtained directly from tissue sections, thereby illustrating how analytes are distributed within these sections. Here we have used an oversampling technique on a commercially available MALDI orthogonal acceleration TOF mass spectrometer with ion mobility separation capability to produce high spatial resolution images of the glycosphingolipid, glucosylceramide (GC). To exemplify the biological application of our approach, GC was imaged in spleen sections from a conditional knockout mouse model of type 1 Gaucher disease in which the catabolism of this glycosphingolipid is impaired. The laser was continually fired at one position until no more ions were observed and then the sample was moved by 15 μm (laser diameter ∼150 μm). Ions were generated from only the unirradiated surface at each of these positions achieving an effective spacing of 15 μm. At 15 μm laser step-size, it was possible to visualize macrophages enriched in GC, which could be distinguished from other cell types in the spleen. Current MALDI MSI spatial resolution is typically limited by the diameter of the laser spot-size, which is usually between 50 and 100 μm, covering an area equivalent to tens of mammalian cells. © 2010 American Chemical Society. Source

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