银杏种子糖蛋白的SDS-PAGE分离及N-糖链的质谱分析
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摘要
利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分离了银杏种子中的糖蛋白组分,进一步用氨水催化释放N-糖链,并采用电喷雾离子质谱(ESI-MS)及在线液相色谱-质谱联用(LC-UV-MS/MS~2)等技术对胶条上释放的N-糖链进行了定性定量分析.结果表明,从银杏种子中分离得到11种糖蛋白,共检测到11条N-糖链,包括高甘露糖型(4. 88%)和复杂型(95. 12%) 2种类型,其中分子量为21000,36000和50000的糖蛋白所释放的核心α-1,3-岩藻糖和β-1,2-木糖修饰的N-糖链所占比率较高,分别为68. 23%,64. 37%和75. 09%.本研究对进一步研究银杏糖蛋白功能具有重要意义.
The glycoproteins in Ginkgo seeds were separated and isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis( SDS-PAGE). The N-glycans released from glycoproteins were qualitatively and quantitatively analyzed by electrospray ionization mass spectrometry( ESI-MS) and on-line liquid chromatography-mass spectrometry( LC-UV-MS/MS~2). As a result,eleven glycoproteins containing oligomannose type glycans( 4. 88%) and complex type glycans( 95. 12%) were obtained. And most of the N-glycans were modified by the core α-1,3-and β-1,2-xylose in the 21000,36000 and 50000 glycoproteins,which accounted for 68. 23%,64. 37% and 75. 09%,respectively. This study can highlight the understanding of protein glycosylation in Ginkgo and provide foundation in the research of Ginkgo biloba seed allergens.
The glycoproteins in Ginkgo seeds were separated and isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis( SDS-PAGE). The N-glycans released from glycoproteins were qualitatively and quantitatively analyzed by electrospray ionization mass spectrometry( ESI-MS) and on-line liquid chromatography-mass spectrometry( LC-UV-MS/MS~2). As a result,eleven glycoproteins containing oligomannose type glycans( 4. 88%) and complex type glycans( 95. 12%) were obtained. And most of the N-glycans were modified by the core α-1,3-and β-1,2-xylose in the 21000,36000 and 50000 glycoproteins,which accounted for 68. 23%,64. 37% and 75. 09%,respectively. This study can highlight the understanding of protein glycosylation in Ginkgo and provide foundation in the research of Ginkgo biloba seed allergens.
引文
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[2] Hart G. W.,Copeland R. J.,Cell,2010,143(5),672—676
[3] Pang P. C.,Chiu P. C.,Lee C. L.,Chang L. Y.,Panico M.,Dell A.,Science,2011,333(6050),1761—1764
[4] Gu J.,Ji T. I.,Xu Q.,Kariya Y.,Gu W.,Fukuda T.,Du Y.,Glycoconjugate J.,2012,29(8),599—607
[5] Altmann F.,Int. Arch. Allergy Imm.,2007,142(2),99—115
[6] Li M.,Jin Y.,Wang X.,Wu Q.,Liu Y.,Li P.,Li X.,Chem. Res. Chinese Universities,2016,32(6),943—946
[7] Bublin M.,Radauer C.,Wilson I. B.,Kraft D.,Scheiner O.,Breiteneder H.,Hoffmann-Sommergruber K.,FASEB J.,2003,17(12),1697—1699
[8] Wilson I. B.,Curr. Opin. Struc. Biol.,2002,12(5),569—577
[9] Du C.,Huang Y.,J. Dermatol. Venereol.,2015,37(5),301—302(杜策,黄莹.皮肤病与性病,2015,37(5),301—302)
[10] Yang J. T.,Wu C. E.,Li Y. Y.,Jia S. Q.,Fan G. J.,Pan H. M.,Agr. Sci. China,2010,43(17),3616—3623(杨剑婷,吴彩娥,李莹莹,贾韶千,范龚健,潘红梅.中国农业科学,2010,43(17),3616—3623)
[11] F9tisch K.,Vieths S.,Glycoconjugate J.,2001,18(5),373—390
[12] Mari A.,Int. Arch. Allergy Imm.,2002,129(4),286—295
[13] Foetisch K.,Westphal S.,Lauer I.,Retzek M.,Altmann F. A.,Kolarich D.,Scheurer S.,Vieths S.,J. Allergy Clin. Immuun.,2003,111(4),889—896
[14] Shi X.,Zhang T.,Li X.,Feng Y.,Tan X.,Jin Y.,Chem. Res. Chinese Universities,2016,32(4),556—560
[15] Paulus K. E.,Mahler V.,Pabst M.,Kogel K. H.,Altmann F.,Sonnewald U.,Front. Plant Sci.,2011,2,42
[16] Okano M.,Kimura Y.,Kino K.,Michigami Y.,Sakamoto S.,Sugata Y.,Maeda M.,Matsuda F.,Kimura M.,Ogawa T.,Nishizaki K.,Clin. Exp. Allergy,2004,34(5),770—778
[17] Maeda M.,Takeda N.,Mano A.,Yamanishi M.,Kimura M.,Kimura Y.,Biosci. Biotech. Bioch.,2013,77(6),1269—1274
[18] Li Y. Y.,Wu C. E.,Yang J. T.,Jia S. Q.,Xu W. B.,Peng F. R.,Food Sci.,2010,31(22),36—40(李莹莹,吴彩娥,杨剑婷,贾韶千,徐文斌,彭方仁.食品科学,2010,31(22),36—40)
[19] Neville Jr D. M.,Glossmann H.,Method Enzymol.,1974,32,92—102
[20] Wang C. J.,Lu Y.,Han J. L.,Jin W. J.,Li L. M.,Zhang Y.,Song X. Z.,Huang L. J.,Wang Z. F.,J. Prothome Res.,2018,17(7),2345—2357
[21] Honda S.,Akao E.,Suzuki S.,Okuda M.,Kakehi K.,Nakamura J.,Anal. Biochem.,1989,180(2),351—357
[22] James A. T.,Yang A.,Food Chem.,2016,194,284—289
[23] Liu C.,Wang H.,Cui Z.,He X.,Wang X.,Zeng X.,Ma H.,Food Chem.,2007,102(4),1310—1316
[24] Li Y. Y.,Wu C. E.,Yang J. T.,Fan G. J.,Food Sci.,2011,32(7),16—20(李莹莹,吴彩娥,杨剑婷,范龚健.食品科学,2011,32(7),16—20)
[25] Aalberse R. C.,Koshte V.,Clemens J. G.,J. Allergy Clin. Immuun.,1981,68(5),356—364
[26] Tretter V.,Altmann F.,Kubelka V.,Int. Arch. Allergy Imm.,1993,102(3),259—266
[27] Ree R. V.,Cabanesmacheteau M.,Akkerdaas J.,J. Biol. Chem.,2000,275(15),11451—11458