Mass Spectrometric Analysis of N-Glycoforms of Soybean Allergenic Glycoproteins Separated by SDS-PAGE.
Glycosylation of many proteins has been revealed to be closely related with food allergies, and screening and structural analysis of related glycoproteins and glycoallergens are essential for studies in this field. Herein, we describe detailed N-glycoform analysis of all glycoprotein fractions of soybean protein isolate (SPI) separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to disclose structural features of the glycan moieties of more soybean glycoproteins. SPI was fractionated by SDS-PAGE, and the generated protein bands were recovered and subjected to in-gel N-glycan release and labeling using a one-pot method newly developed by our group, followed by detailed analysis by electrospray ionization mass spectrometry (ESI-MS) and online hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HILIC-ESI-MS/MS). As a result, we found seven bands mainly containing oligomannose-type glycans; two mainly contain core α1,3-fucosylated glycans, and six have no glycans. This study is the first report that discovers core α1,3-fucosylated N-glycans in bands 1, 2, and 6 and discloses bands 3, 4, 5, and 7 as glycoproteins and their N-glycoforms. Therefore, it can expand our knowledge about soybean protein glycosylation and provide significant structural reference for research of soybean allergens.
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education and Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University , Xi'an 710069, China.
Lingmei
L
Li
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education and Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University , Xi'an 710069, China.
Chengjian
C
Wang
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education and Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University , Xi'an 710069, China.
Shan
S
Qiang
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education and Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University , Xi'an 710069, China.
Jixiang
J
Zhao
School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University , Dalian 116034, China.
Shuang
S
Song
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education and Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University , Xi'an 710069, China.
Wanjun
W
Jin
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education and Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University , Xi'an 710069, China.
Bo
B
Wang
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education and Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University , Xi'an 710069, China.
Ying
Y
Zhang
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education and Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University , Xi'an 710069, China.
Linjuan
L
Huang
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education and Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University , Xi'an 710069, China.
Zhongfu
Z
Wang
D000485:Allergens / Q000737:chemistry*; D052179:Antigens, Plant / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D011134:Polysaccharides / Q000737:chemistry; D013025:Glycine max / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D000485:Allergens; D052179:Antigens, Plant; D006023:Glycoproteins; D011134:Polysaccharides
ESI-MS; HILIC-ESI-MS/MS; N-glycans; glycoprotein; soybean allergy