title: Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid.
abstract: Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.
authors: Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan. w-makoto@shimadzu.co.jp
authors: Makoto
authors: M
authors: Watanabe
authors: Kazuya
authors: K
authors: Terasawa
authors: Kaoru
authors: K
authors: Kaneshiro
authors: Hiromasa
authors: H
authors: Uchimura
authors: Rie
authors: R
authors: Yamamoto
authors: Yuko
authors: Y
authors: Fukuyama
authors: Kazuharu
authors: K
authors: Shimizu
authors: Taka-Aki
authors: TA
authors: Sato
authors: Koichi
authors: K
authors: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D000634:Aminoquinolines / Q000737:chemistry; D002240:Carbohydrate Sequence; D002460:Cell Line; D003373:Coumaric Acids / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000634:Aminoquinolines; D003373:Coumaric Acids; D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides; D018719:Erb-b2 Receptor Tyrosine Kinases; C007175:alpha-cyano-4-hydroxycinnamate; C508053:ERBB2 protein, human