title: Development of quantitative plasma N-glycoproteomics using label-free 2-D LC-MALDI MS and its applicability for biomarker discovery in hepatocellular carcinoma.
abstract: There has been rapid progress in the development of clinical proteomic methodologies with improvements in mass spectrometric technologies and bioinformatics, leading to many new methodologies for biomarker discovery from human plasma. However, it is not easy to find new biomarkers because of the wide dynamic range of plasma proteins and the need for their quantification. Here, we report a new methodology for relative quantitative proteomic analysis combining large-scale glycoproteomics with label-free 2-D LC-MALDI MS. In this method, enrichment of glycopeptides using hydrazide resin enables focusing on plasma proteins with lower abundance corresponding to the tissue leakage region. On quantitative analysis, signal intensities by 2-D LC-MALDI MS were normalized using a peptide internal control, and the values linked to LC data were treated with DeView™ software. Our proteomic method revealed that the quantitative dynamic ranged from 10² to 10⁶ pg/mL of plasma proteins with good reproducibility, and the limit of detection was of the order of a few ng/mL of proteins in biological samples. To evaluate the applicability of our method for biomarker discovery, we performed a feasibility study using plasma samples from patients with hepatocellular carcinoma, and identified biomarker candidates, including ceruloplasmin, alpha-1 antichymotrypsin, and multimerin-1.
authors.affiliation: Discovery Research Laboratories, Shionogi & Co. Ltd., Fukushima-ku, Osaka 553-0002, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ishihara
authors.forename: Isao
authors.initials: I
authors.lastname: Fukuda
authors.forename: Atsushi
authors.initials: A
authors.lastname: Morita
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Takinami
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Okamoto
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Numata
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood*; D006528:Carcinoma, Hepatocellular / Q000097:blood*; D002570:Ceruloplasmin / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D005240:Feasibility Studies; D006020:Glycopeptides / Q000097:blood*; D006801:Humans; D008113:Liver Neoplasms / Q000097:blood*; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D000514:alpha 1-Antichymotrypsin / Q000097:blood
chemical_list: D014408:Biomarkers, Tumor; D006020:Glycopeptides; D000514:alpha 1-Antichymotrypsin; D002570:Ceruloplasmin