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Richard Barry

Publications and source records attributed to Richard Barry.

8 recordsLinked to original sources
PubMed2005

Transmembrane adaptor molecules: a new category of lymphoid-cell markers.

Transmembrane adaptor proteins (of which 7 have been identified so far) are involved in receptor signaling in immune cells. They have only a short extracellular region, with most of the molecule comprising a substantial intracytoplasmic region carrying multiple tyrosine residues that can be phosphorylated by Src- or Syk-family kinases. In this paper, we report an immunohistologic study of 6 of these molecules in normal and neoplastic human tissue sections and show that they are restricted to subpopulations of lymphoid cells, being present in either T cells (LAT, LIME, and TRIM), B cells (NTAL), or subsets of both cell types (PAG and SIT). Their expression in neoplastic lymphoid cells broadly reflects that of normal lymphoid tissue, including the positivity of plasma cells and myeloma/plasmacytoma for LIME, NTAL, PAG, and SIT. However, this study also revealed some reactions that may be of diagnostic/prognostic value. For example, lymphocytic lymphoma and mantle-cell lymphoma showed similar profiles but differed clearly from follicle-center lymphoma, whereas PAG tended to be selectively expressed in germinal center-derived subsets of diffuse large B-cell lymphoma. These molecules represent a potentially important addition to the panel of immunophenotypic markers detectable in routine biopsies that can be used in hematopathologic studies.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Microfluidics in biotechnology.

Microfluidics enables biotechnological processes to proceed on a scale (microns) at which physical processes such as osmotic movement, electrophoretic-motility and surface interactions become enhanced. At the microscale sample volumes and assay times are reduced, and procedural costs are lowered. The versatility of microfluidic devices allows interfacing with current methods and technologies. Microfluidics has been applied to DNA analysis methods and shown to accelerate DNA microarray assay hybridisation times. The linking of microfluidics to protein analysis techologies, e.g. mass spectrometry, enables picomole amounts of peptide to be analysed within a controlled micro-environment. The flexibility of microfluidics will facilitate its exploitation in assay development across multiple biotechnological disciplines.

Journal Article↗
PubMed2004

Quantitative protein profiling using antibody arrays.

Traditional approaches to microarrays rely on direct binding assays where the extent of hybridisation and the signal detected are a measure of the analyte concentration in the experimental sample. This approach, directly imported from the nucleic acid field, may fail if applied to antibody-antigen interactions due to the shortage of characterised antibodies, the significant heterogeneity of antibody affinities, their dependence on the extent of protein modification during labelling and the inherent antibody cross-reactivity. These problems can potentially limit the multiplexing capabilities of protein affinity assays and in many cases rule out quantitative protein profiling using antibody microarrays. A number of approaches aimed at achieving quantitative protein profiling in a multiplex format have been reported recently. Of those reported, the three most promising routes include signal amplification, multicolour detection and competitive displacement approaches to multiplex affinity assays. One in particular, competitive displacement, also overcomes the problems associated with quantitation of affinity interactions and provides the most generic approach to highly parallel affinity assays, including antibody arrays.

Animals↗
PubMed2003

Combinatorial peptidomics: a generic approach for protein expression profiling.

Traditional approaches to protein profiling were built around the concept of investigating one protein at a time and have long since reached their limits of throughput. Here we present a completely new approach for comprehensive compositional analysis of complex protein mixtures, capable of overcoming the deficiencies of current proteomics techniques. The Combinatorial methodology utilises the peptidomics approach, in which protein samples are proteolytically digested using one or a combination of proteases prior to any assay being carried out. The second fundamental principle is the combinatorial depletion of the crude protein digest (i.e. of the peptide pool) by chemical crosslinking through amino acid side chains. Our approach relies on the chemical reactivities of the amino acids and therefore the amino acid content of the peptides (i.e. their information content) rather than their physical properties. Combinatorial peptidomics does not use affinity reagents and relies on neither chromatography nor electrophoretic separation techniques. It is the first generic methodology applicable to protein expression profiling, that is independent of the physical properties of proteins and does not require any prior knowledge of the proteins. Alternatively, a specific combinatorial strategy may be designed to analyse a particular known protein on the basis of that protein sequence alone or, in the absence of reliable protein sequence, even the predicted amino acid translation of an EST sequence. Combinatorial peptidomics is especially suitable for use with high throughput micro- and nano-fluidic platforms capable of running multiple depletion reactions in a single disposable chip.

Journal Article↗
PubMed2003

Peptidomics: A new approach to affinity protein microarrays.

Protein microarrays for diagnostic and proteomic analyses are being developed using a number of different techniques for each of the steps required including immobilisation methods, assay and detection systems. This is extremely different to the development of DNA microarrays which is now a well established technology that has demonstrated the capabilities of transcriptomics to deliver validated differential transcripts. As mRNA and protein levels do not always correlate, protein microarrays would seem to be an obvious successor to DNA arrays. Unlike nucleic acids, however, protein targets are typically nonhomogeneous in physicochemical properties and affinity capture agents are often poorly characterised making the experiments difficult to perfect and reproduce. Moreover, running multiple affinity assays in parallel (multiplexing) is compromised by the heterogeneity of antibody affinities to their protein targets. In the peptidomic approach presented here the assayed mixture of proteins is enzymatically digested prior to affinity capture to form a mixture of short peptides that are more similar in their physicochemical properties than intact proteins. These peptides can be predicted by in silico digestion of individual proteins, e.g. from protein databases allowing design of nonhomologous reagents for the screening of affinity agent libraries. The use of mass spectrometry (e.g. matrix-assisted laser desorption/ionization-time of flight mass spectrometry) for a direct confirmation of the identity of the species captured, provides a further advantage compared to the more usual method of detection in which fluorescently labelled captured species are scanned to give a spatially resolved image of the array.

Peptides↗
PubMed2003

Competitive assay formats for high-throughput affinity arrays.

The authors describe a novel method for the quantitation of differential levels of biomolecules using unlabeled samples and protein-binding arrays for assessing differential expression. Traditional affinity arrays, whether in microplates or protein microarrays, suffer from a few common problems-a shortage of characterized antibodies and highly variable affinities for those available. Also, the assayed proteins could be present in a wide range of concentrations and physicochemical properties, so that it becomes an onerous task to optimize assay conditions for each antibody-antigen pair. Currently, this restricts parallel affinity assays to a low number of carefully selected antibodies and restricts the development of highly multiplexed parallel affinity assays. A displacement strategy allows the use of a much wider range of antibodies, reducing the requirement for matched affinities. The competitive assays described here also show a much higher tolerance for nonspecific background noise. The range of assayed protein concentrations is only limited by the sensitivity of the detection system used.

Antibody Affinity↗
PubMed2002

Reproducibility of targeted gene expression measurements in human islets of Langerhans.

The expression of 47 genes involved in the biosynthesis and secretion of insulin, apoptosis, and cellular stress was evaluated in isolated human islets using cDNA probes arrayed on nitrocellulose membranes. Isolated human islets were cultured for four days, or one month, with glucose present at a concentration of either 5.5 or 16.7 mmol/L. Extracted islet total RNA was used to generate [32P]dATP-labelled complex cDNA targets and hybridised with immobilised cDNA arrays. The positive expression of 45 mRNA transcripts in isolated human islets was documented. The coefficient of variance for relative levels of expression of transcripts was <25% for 9, 25-50% for 22, and 50-100% for 10, indicating good reproducibility between islet preparations from five different human pancreas donors. This study demonstrates the utility of nitrocellulose-based cDNA arrays for a focused reproducible analysis of gene expression changes in human islets of Langerhans.

Cells, Cultured↗