Lectin-Based SP3 Technology Enables N-Glycoproteomic Analysis of Mouse Oocytes

凝集素 计算生物学 细胞生物学 化学 生物 生物化学
作者
Zian Huo,Haixia Tu,Jie Ren,Xiangzheng Zhang,Yaling Qi,Chenghao Situ,Yan Li,Yueshuai Guo,Xuejiang Guo,Hui Zhu
出处
期刊:Journal of Proteome Research [American Chemical Society]
卷期号:23 (6): 2137-2147
标识
DOI:10.1021/acs.jproteome.4c00089
摘要

N-glycosylation is one of the most universal and complex protein post-translational modifications (PTMs), and it is involved in many physiological and pathological activities. Owing to the low abundance of N-glycoproteins, enrichment of N-glycopeptides for mass spectrometry analysis usually requires a large amount of peptides. Additionally, oocyte protein N-glycosylation has not been systemically characterized due to the limited sample amount. Here, we developed a glycosylation enrichment method based on lectin and a single-pot, solid-phase-enhanced sample preparation (SP3) technology, termed lectin-based SP3 technology (LectinSP3). LectinSP3 immobilized lectin on the SP3 beads for N-glycopeptide enrichment. It could identify over 1100 N-glycosylation sites and 600 N-glycoproteins from 10 μg of mouse testis peptides. Furthermore, using the LectinSP3 method, we characterized the N-glycoproteome of 1000 mouse oocytes in three replicates and identified a total of 363 N-glycosylation sites from 215 N-glycoproteins. Bioinformatics analysis revealed that these oocyte N-glycoproteins were mainly enriched in cell adhesion, fertilization, and sperm-egg recognition. Overall, the LectinSP3 method has all procedures performed in one tube, using magnetic beads. It is suitable for analysis of a low amount of samples and is expected to be easily adaptable for automation. In addition, our mouse oocyte protein N-glycosylation profiling could help further characterize the regulation of oocyte functions.
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