title: A lectin-coupled, multiple reaction monitoring based quantitative analysis of human plasma glycoproteins by mass spectrometry.
abstract: Aberrant protein glycosylation may be closely associated with cancer pathology. To measure the abundance of protein glycoforms with a specific glycan structure in plasma samples, we developed a lectin-coupled multiple reaction monitoring (MRM)-based mass spectrometric method. It was confirmed that the method could provide reproducible results with precision sufficient to distinguish differences in the abundance of protein glycoforms between individuals. Plasma samples prepared from hepatocellular carcinoma (HCC) patients without immuno-depletion of highly abundant plasma proteins were fractionated by use of fucose-specific aleuria aurantia lectin (AAL) immobilized on magnetic beads by use of a biotin-streptavidin conjugate. The lectin-captured fractions were digested by trypsin and profiled by tandem mass spectrometry. From the proteomic profiling data, target glycoproteins were selected and analyzed quantitatively by MRM-based analysis. The reproducibility of MRM-based quantification of the selected target proteins was reliable, with precision (CV; ≤14% for batch-to-batch replicates and ≤19% for replicates over three days) sufficient to distinguish differences in the abundance of AAL-captured glycoforms between individual plasma samples. This lectin-coupled, MRM-based method, measuring only lectin-captured glycoforms of a target protein rather than total target protein, is a tool for monitoring differences between individuals by measuring the abundance of aberrant glycoforms of a target protein related to a disease. This method may be further applied to rapid verification of biomarker candidates involved in aberrant protein glycosylation in human plasma.
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, Ochang-Eup, Cheongwon-Gun, Republic of Korea.
authors.forename: Yeong Hee
authors.initials: YH
authors.lastname: Ahn
authors.forename: Park Min
authors.initials: PM
authors.lastname: Shin
authors.forename: Eun Sun
authors.initials: ES
authors.lastname: Ji
authors.forename: Hoguen
authors.initials: H
authors.lastname: Kim
authors.forename: Jong Shin
authors.initials: JS
authors.lastname: Yoo
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000737:chemistry; D006528:Carcinoma, Hepatocellular / Q000097:blood; D005656:Fungal Proteins / Q000737:chemistry; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D037102:Lectins / Q000737:chemistry; D008113:Liver Neoplasms / Q000097:blood; D013058:Mass Spectrometry / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D005656:Fungal Proteins; D006023:Glycoproteins; D055767:Immobilized Proteins; D037102:Lectins; C064549:lectin, Aleuria aurantia