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One-Step Labeling Strategy for the Profiling of Multiple Types of Protein Glycosylation.

Yinping Tian,Yuqiu Wang,Ying Zhang,Jingyi Guo,Pengfei Zhang,Xia Li,Huancheng Zhou,L. Wen

Published 2025 in Analytical Chemistry

ABSTRACT

Protein glycosylation, classified into N-glycosylation and O-glycosylation, is the most prevalent and complex protein post-translational modification. Bioorthogonal chemistry reactions combining the biotin-streptavidin interaction system are the most commonly used for investigating protein glycosylation. In this study, a one-step enzymatic labeling strategy for the simultaneous and global profiling of multiple types of protein glycosylation was developed. A "one-step probe" directly carrying the enrichment support poly(N-isopropylacrylamide) (PNIPAM) was designed and synthesized. Although the designed probe carried a large enrichment group (the number-average molecular weight of PNIPAM was up to 10,000 Da), it was well accepted by the two substrate-specific sialyltransferases to label N-glycopeptides and O-glycopeptides. PNIPAM is a temperature-sensitive polymer. When the temperature was below the lower critical solution temperature, PNIPAM was water-soluble and precipitated when it was above the lower critical solution temperature. The advantage of this property was that the labeled glycopeptides were enriched from complex biological samples by simply changing the temperature without the need for additional enrichment resins. Following enzymatic and ultraviolet-light-mediated cleavage, the labeled N-glycopeptide, core-fucosylated glycopeptide, and truncated mucin-type O-glycopeptides (Tn, STn, T, and ST antigens) were released sequentially for glycosylation profiling via mass spectrometry. This work provides an effective strategy to significantly reduce enrichment costs for profiling multiple glycosylation types.

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