Highly Efficient Analysis of Glycoprotein Sialylation in Human Serum by Simultaneous Quantification of Glycosites and Site-specific Glycoforms.

Aberrant sialylation of glycoproteins is closely related to many malignant diseases, and analysis of sialylation has great potential to reveal the status of those diseases. However, it is still challenging to in-depth analyse the sialylation, due to the high micro-heterogeneity of protein glycosylation, as well as the low abundance of sialylated glycopeptides (SGPs). Herein, an integrated strategy was fabricated for the detail characterization of glycoprotein sialylation on glycosite and site-specific glycoform levels by employing the SGP enrichment method. This strategy enabled the identification of up to 380 glycosites, as well as 414 intact glycopeptides corresponding to 383 site-specific glycoforms from only initial 6 μL serum samples, indicating the high sensitivity of the method for detail analysis of glycoprotein sialylation. This strategy was further employed to differential analysis of glycoprotein sialylation between hepatocellular carcinoma (HCC) patients and control samples, leading to quantification of 344 glycosites and 405 site-specific glycoforms, simultaneously. Among them, 43 glycosites and 55 site-specific glycoforms were found to have significant change on the glycosite and site-specific glycoform levels, respectively. Interestingly, several glycoforms on the same glycosite were found with different change tendency. This strategy was demonstrated to be a powerful tool to reveal subtle difference of macro- and micro-heterogeneity of glycoprotein sialylation.

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