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

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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 ongoing efforts in glyco-biomarker discovery.

Original languageEnglish
Pages (from-to)99-108
Number of pages10
JournalMolecular and Cellular Proteomics
Volume8
Issue number1
DOIs
Publication statusPublished - 2009 Jan 1

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ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

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