The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.
Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.
Sun
Xiuxuan
X
College of Life Sciences, Northwest University, 710069 Xi'an, P. R. China.
Yang
Ganglong
G
Sun
Shisheng
S
Quan
Rui
R
Dai
Weiwei
W
Li
Bin
B
Chen
Chao
C
Li
Zheng
Z
D002236:Carbohydrate Conformation; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006878:Hydroxides / Q000737:chemistry*; D008024:Ligands; D008280:Magnetics; D010003:Osteoarthritis / Q000097:blood; D011134:Polysaccharides / Q000097:blood / Q000302:isolation & purification*; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification*; D017550:Spectroscopy, Fourier Transform Infrared; D013499:Surface Properties; D013577:Syndrome
D006878:Hydroxides; D008024:Ligands; D011134:Polysaccharides; D011506:Proteins