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 beta(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 with invasive ductal breast carcinoma. Comparison analysis of the data identified 34 proteins that were enriched by L-PHA fractionation in tumor relative to normal tissue for at least 2 cases of ductal invasive breast carcinoma. Of these 34 L-PHA tumor enriched proteins, 12 are common to all 4 matched cases analyzed. These results indicate that lectin enrichment strategies targeting a particular glycan change associated with malignancy can be an effective method of identifying potential biomarkers for breast carcinoma.

Focused Differential Glycan Analysis with the Platform Antibody-assisted Lectin Profiling for Glycan-related Biomarker Verification

“…Recent advances in glycoproteomics produced a number of glycoproteins, which have been nominated as disease-related biomarker candidates (14,(43)(44)(45)(46)(47). As a standard procedure employed in concurrent proteomics-based biomarker development, the candidates are first quantified with the samples used in the candidate identification phase, then verified via a hundred clinical samples before the last validation phase (10).…”

Developmental Expression of the Neuron-specific N-Acetylglucosaminyltransferase Vb (GnT-Vb/IX) and Identification of Its in Vivo Glycan Products in Comparison with Those of Its Paralog, GnT-V

“…All brains for this study were immediately frozen at Ϫ80°C until glycomic analysis. The frozen whole brains were homogenized and delipidated by extraction twice for 3 h at room temperature on the rocker with a mixture of chloroform/methanol/water (4:8:3, v/v/v) as described (54,55). The emulsion was centrifuged at 2,800 ϫ g for 15 min at 4°C to remove the supernatant.…”

The galectin lattice at a glance

“…Galectins bind to glycan motifs found on many glycoconjugates at the cell surface and, in this regard, might appear to lack specificity for individual effectors. Indeed, proteomic analyses of Gal3-interacting partners, Mgat5-dependent raft association and L-PHA-binding proteins (the higher affinity galectin ligands) indicate binding of many glycoproteins in parallel (Abbott et al, 2008;Boscher et al, 2012;Lakshminarayan et al, 2014). Nonetheless, a systems-based approach has revealed a selective association of transmembrane glycoproteins with the galectin lattice .…”