An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

被引:40
作者
Chen, Minyong [1 ]
Shi, Xiaofeng [1 ]
Duke, Rebecca M. [1 ]
Ruse, Cristian I. [1 ]
Dai, Nan [1 ]
Taron, Christopher H. [1 ]
Samuelson, James C. [1 ]
机构
[1] New England Biolabs Inc, 240 Cty Rd, Ipswich, MA 01938 USA
关键词
MASS-SPECTROMETRY; UBIQUITIN LIGASES; GLYCOPROTEIN ENRICHMENT; COMPREHENSIVE ANALYSIS; BIOMARKER DISCOVERY; STRUCTURAL BASIS; HUMAN PLASMA; GLYCOSYLATION; LECTIN; CANCER;
D O I
10.1038/ncomms15487
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
A method for selective and comprehensive enrichment of N-linked glycopeptides was developed to facilitate detection of micro-heterogeneity of N-glycosylation. The method takes advantage of the inherent properties of Fbs1, which functions within the ubiquitin-mediated degradation system to recognize the common core pentasaccharide motif (Man3GlcNAc2) of N-linked glycoproteins. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules; however, wild-type Fbs1 preferentially binds high-mannose-containing glycans. We identified Fbs1 variants through mutagenesis and plasmid display selection, which possess higher affinity and improved recovery of complex N-glycomolecules. In particular, we demonstrate that the Fbs1 GYR variant may be employed for substantially unbiased enrichment of N-linked glycopeptides from human serum. Most importantly, this highly efficient N-glycopeptide enrichment method enables the simultaneous determination of N-glycan composition and N-glycosites with a deeper coverage (compared to lectin enrichment) and improves large-scale N-glycoproteomics studies due to greatly reduced sample complexity.
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页数:15
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