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

Research output: Contribution to journalArticle

89 Citations (Scopus)

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
Externally publishedYes

Fingerprint

Biomarkers
Lectins
Polysaccharides
Glycosylation
Glycoproteins
Antibodies
Microarrays
Proteins
Tissue Extracts
Proteome
Prostate-Specific Antigen
Immunoprecipitation
Immunoblotting
Proteomics
Pipelines
Throughput
Tissue
Molecules
Serum

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Cite this

Focused differential glycan analysis with the platform antibody-assisted lectin profiling for glycan-related biomarker verification. / Kuno, Atsushi; Kato, Yukinari; Matsuda, Atsushi; Kaneko, Mika Kato; Ito, Hiromi; Amano, Koh; Chiba, Yasunori; Narimatsu, Hisashi; Hirabayashi, Jun.

In: Molecular and Cellular Proteomics, Vol. 8, No. 1, 01.2009, p. 99-108.

Research output: Contribution to journalArticle

Kuno, Atsushi ; Kato, Yukinari ; Matsuda, Atsushi ; Kaneko, Mika Kato ; Ito, Hiromi ; Amano, Koh ; Chiba, Yasunori ; Narimatsu, Hisashi ; Hirabayashi, Jun. / Focused differential glycan analysis with the platform antibody-assisted lectin profiling for glycan-related biomarker verification. In: Molecular and Cellular Proteomics. 2009 ; Vol. 8, No. 1. pp. 99-108.
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