Focused differential glycan analysis with the platform antibody-assisted lectin profiling for glycan-related biomarker verification.
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.
Kuno
Atsushi
A
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Central 2, 1-1-1, Umezono, Ibaraki 305-8568, Japan.
Kato
Yukinari
Y
Matsuda
Atsushi
A
Kaneko
Mika Kato
MK
Ito
Hiromi
H
Amano
Koh
K
Chiba
Yasunori
Y
Narimatsu
Hisashi
H
Hirabayashi
Jun
J
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D015415:Biomarkers / Q000032:analysis*; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000032:analysis*; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D010974:Platelet Aggregation; D011134:Polysaccharides / Q000032:analysis*; D017430:Prostate-Specific Antigen / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D051381:Rats; D015203:Reproducibility of Results; D014020:Tissue Extracts; D000098853:Podoplanin
D000906:Antibodies; D015415:Biomarkers; D037102:Lectins; D008562:Membrane Glycoproteins; D019158:N-Acetylneuraminic Acid; D011134:Polysaccharides; D017430:Prostate-Specific Antigen; D014020:Tissue Extracts; C117468:PDPN protein, human; D000098853:Podoplanin