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

Atsushi Kuno,Yukinari Kato,Atsushi Matsuda,Mika Kato Kaneko,Hiromi Ito,Koh Amano,Yasunori Chiba,Hisashi Narimatsu,Jun Hirabayashi

doi:10.1074/mcp.m800308-mcp200

Abstract

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 high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.

Highlights

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
A specific glycan profile of a target glycoprotein is acquired with the aid of a specific antibody raised against the core protein moiety, removing the need to covalently label each glycoprotein with a fluorescent reagent (Refs. 22–29 and Fig. 1A)
There is a critical issue to be overcome when using antibodies for glycoprotein detection, since the antibody glycans potentially interact with lectins immobilized on the glass slide (Fig. 2A, top)

Summary

Differential Glycan Profiling for Biomarker Verification

Assays with more than 30 lectins to search for critical differences in glycan structures. “antibody-overlay lectin microarray” [21], was taken, which is based on the lectin microarray platform originally developed in our laboratory [22,23,24]. In this strategy, a specific glycan profile of a target glycoprotein is acquired with the aid of a specific antibody raised against the core protein moiety, removing the need to covalently label each glycoprotein with a fluorescent reagent Once a target glycoprotein (Ͻ100 ng) is enriched from a trace amount of crude samples by a microliter-scale immunoprecipitation procedure with the biotinylated antibody used for the above detection, a precise glycan profile of the target protein is obtained in a highly reproducible and high-throughput manner (see Fig. 1B). The system provides a feasible and powerful advance toward “focused differential glycoproteomics”

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION