Showing papers by "Lance Wells published in 2008"

Targeted glycoproteomic identification of biomarkers for human breast carcinoma.

Abstract: Glycosylation is a dynamic post-translational modification that changes during the development and progression of various malignancies. During the oncogenesis of breast carcinoma, the glycosyltransferase known as N-acetylglucosaminyltransferase Va (GnT-Va) transcript levels and activity are increased due to activated oncogenic signaling pathways. Elevated GnT-V levels leads to increased β(1,6)-branched N-linked glycan structures on glycoproteins that can be measured using a specific carbohydrate binding protein or lectin known as L-PHA. L-PHA does not bind to nondiseased breast epithelial cells, but during the progression to invasive carcinoma, cells show a progressive increase in L-PHA binding. We have developed a procedure for intact protein L-PHA-affinity enrichment, followed by nanospray ionization mass spectrometry (NSI-MS/MS), to identify potential biomarkers for breast carcinoma. We identified L-PHA reactive glycoproteins from matched normal (nondiseased) and malignant tissue isolated from patients...

Defining the regulated secreted proteome of rodent adipocytes upon the induction of insulin resistance.

Abstract: Insulin resistance defines the metabolic syndrome and precedes, as well is the hallmark of, type II diabetes. Adipocytes, besides being a major site for energy storage, are endocrine in nature and secrete a variety of proteins, adipocytokines (adipokines), that can modulate insulin sensitivity, inflammation, obesity, hypertension, food intake (anorexigenic and orexigenic), and general energy homeostasis. Recent data demonstrates that increased intracellular glycosylation of proteins via O-GlcNAc can induce insulin resistance and that a rodent model with genetically elevated O-GlcNAc levels in muscle and fat displays hyperleptinemia. The link between O-GlcNAc levels, insulin resistance, and adipocytokine secretion is further explored here. First, with the use of immortalized and primary rodent adipocytes, the secreted proteome of differentiated adipocytes is more fully elucidated by the identification of 97 and 203 secreted proteins, respectively. Mapping of more than 80 N-linked glycosylation sites on adipocytokines from the cell lines further defines this proteome. Importantly, adipocytokines that are modulated when cells are shifted from insulin responsive to insulin resistant conditions are determined. By the use of two protocols for inducing insulin resistance, classical hyperglycemia with chronic insulin exposure and pharmacological elevation of O-GlcNAc levels, several proteins are identified that are regulated in a similar fashion under both conditions including HCNP, Quiescin Q6, Angiotensin, lipoprotein lipase, matrix metalloproteinase 2, and slit homologue 3. Detection of these potential prognostic/diagnostic biomarkers for metabolic syndrome, type II diabetes, and the resulting complications of both diseases further establishes the central role of the O-GlcNAc modification of intracellular proteins in the pathophysiology of these conditions.

Use of cancer-specific yeast-secreted in vivo biotinylated recombinant antibodies for serum biomarker discovery

Abstract: Strategies to discover circulating protein markers of ovarian cancer are urgently needed. We developed a novel technology that permits us to isolate recombinant antibodies directed against the potential serum biomarkers, to facilitate the further development of affinity reagents necessary to construct diagnostic tests. This study presents a novel discovery approach based on serum immunoprecipitation with cancer-specific in vivo biotinylated recombinant antibodies (biobodies) derived from differentially selected yeast-display scFv, and analysis of the eluted serum proteins by electrophoresis and/or mass spectrometry. Using this strategy we identified catabolic fragments of complement factors, EMILIN2, Von Willebrand factor and phosphatidylethanolamine-binding protein 1 (PEBP1 or RKIP) in patient sera. To our knowledge, this is the first report of a soluble form of PEBP1 in human. Independent evidence for ovarian cancer-specific expression of PEBP1 in patient sera was found by ELISA assays and antibody arrays with anti-PEBP1 antibodies. PEBP1 was detected in 29 out of 30 ascites samples and discriminated ovarian cancer sera from controls (p = 0.02). Finally, we confirmed by western blots the presence of a 21–23 kDa fragment corresponding to the expected size of PEBP1 but we also showed additional bands of 38 kDa and 50–52 kDa in various tissues and cell lines. We conclude that the novel strategy described here allows the identification of candidate biomarkers that can be variants of normally expressed proteins or that display cancer-specific post-translational modifications.

Chapter 15 O-GlcNAc Proteomics: Mass Spectrometric Analysis of O-GlcNAc Modifications on Proteins

Abstract: Publisher Summary This chapter reviews the specific functions of O-linked N-acetylglucosamine (O-GlcNAc) to establish its biological significance. The reason this modification has been historically difficult to detect is provided and the recent efforts to develop methods/techniques allowing for effective mass spectrometry (MS)-based site-specific O-GlcNAc proteomics are compared. O-GlcNAcylation is a dynamic enzyme-mediated cytosolic and nuclear carbohydrate modification of serines and threonines by NAc that is in many ways analogous to phosphorylation. One of the key features of O-GlcNAcylation that sets it apart from complex glycosylation is that it rapidly turns over on proteins and is responsive to specific cellular signals and changing cell states. The pharmacological agents—calcium ionophore and okadaic acid—that bypass agonist-induced signals to effect calcium- and phosphorylation-based signaling cause O-GlcNAc responsiveness within one minute, thereby demonstrating the potential for rapid signaling-based modulation of O-GlcNAc modification. In response to a variety of cellular stresses, a complex protective adaptive program has been initiated in eukaryotic cells, which is associated with rapidly elevated O-GlcNAc modification levels.