Takahashi Kazuo, Hiki Yoshiyuki, Odani Hiroko, Shimozato Sachiko, Iwase Hitoo, Sugiyama Satoshi, Usuda Nobuteru
Department of Anatomy, Fujita Health University, School of Medicine, 1-98 Denngakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan.
Biochem Biophys Res Commun. 2006 Nov 24;350(3):580-7. doi: 10.1016/j.bbrc.2006.09.075. Epub 2006 Sep 25.
The aim of the study was to develop a simple and precise method for identifying glycosylation of the IgA hinge region using surface-enhanced laser desorption/ionization (SELDI)-TOFMS with a lectin-coupled ProteinChip array. Serum IgA was isolated using an anti-IgA antibody column. Following reduction, alkylation, and trypsin digestion, the IgA fragments were applied on the ProteinChip coupled with jacalin, peanut agglutinin (PNA), or Vilsa villosa lectin (VVL). The SELDI-TOFMS peaks corresponding to the fragments containing IgA1 hinge glycopeptides trapped by each lectin were compared. The jacalin-, PNA-, and VVL-immobilized ProteinChips detected 13, 4, and 2 peaks, respectively. One major peak was confirmed as a glycopeptide by MS/MS analysis. These results suggest that a lectin-immobilized ProteinChip assay can be used to simplify the procedures for the analyses of the O-glycans in IgA1 hinge. This method potentially makes it possible to identify a disease-specific glycoform by selecting the appropriate ligand-coupled ProteinChip array.
本研究的目的是开发一种简单而精确的方法，使用凝集素偶联的蛋白质芯片阵列通过表面增强激光解吸/电离（SELDI）-TOFMS来鉴定IgA铰链区的糖基化。使用抗IgA抗体柱分离血清IgA。经过还原、烷基化和胰蛋白酶消化后，将IgA片段应用于偶联了红豆凝集素、花生凝集素（PNA）或绒毛豆凝集素（VVL）的蛋白质芯片上。比较了与每种凝集素捕获的含有IgA1铰链糖肽的片段相对应的SELDI-TOFMS峰。偶联红豆凝集素、PNA和VVL的蛋白质芯片分别检测到13个、4个和2个峰。通过串联质谱分析确认一个主要峰为糖肽。这些结果表明，凝集素偶联的蛋白质芯片分析可用于简化IgA1铰链中O-聚糖的分析程序。该方法有可能通过选择合适的配体偶联蛋白质芯片阵列来鉴定疾病特异性糖型。
