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

2009, Molecular & Cellular Proteomics

Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and highthroughput manner. Thus, the established system should provide a powerful pipeline in support of ongoing efforts in glyco-biomarker discovery. Molecular & Cellular Proteomics 8:99-108, 2009. Glycan synthesis in individual cells is regulated by harmonized expression of more than a hundred glycosyltransferases. Importantly, detectable dynamics occurring on each cell surface during diverse biological events, e.g. differentiation, proliferation, and signal induction, indicate drastic changes in the glyco-machinery (1). During the last few decades, there have been enormous advances in the findings of glycosylation alterations related to oncogenesis. Cell surface sialylation and ␤1-6 branching of N-linked glycans are strongly correlated with metastatic potential of cancer cells (2, 3). Metastatic ability is also reflected by dramatic alteration of core structures of O-glycans (4). These observations suggest that novel tumor-specific glycoproteins accompanying substantial structural changes in glycan moieties will become reliable biomarkers with higher specificity than those established previously, e.g. CA19-9 (5), carcino-embryonic antigen family (6), ␣-fetoprotein (AFP) 1 (7), and prostate-specific antigen (PSA) (8). Recent advances in technologies such as mass spectrometry (MS), microarray, and laser microdissection have strongly advanced the proteomics-based biomarker discovery phase targeting both cultured cells and tissue specimens (9-14). This has resulted in the emergence of an extensive range of biomarker "candidates", many of which are glycoproteins. However, these candidate molecules need to be subsequently subjected to a verification step prior to a much large scale validation phase (e.g. treating with Ͼ1,000 incidences). Antibody microarray has taken the place of ELISA as a more versatile and high-throughput technique, and has enabled multiplexed quantitative analysis for over a hundred proteins with sufficient sensitivity (15). As an application of MS technology, multiple-reaction monitoring-MS has also been developed (16-19). However, taking into consideration the critical glycosylation changes occurring on diverse glycoproteins, differential analysis of respective glycans is necessary in parallel with quantitative protein analysis. In this context, Chen et al. (20) have recently developed a lectin-overlay antibody microarray with the intention of discovering glycoprotein biomarkers. However, the proposed strategy requires repeated