Separation And Purification Of Sialylglycopeptide From Egg Yolk Based On Cotton Hydrophilic Chromatography

The peptide portion of the sialylglycopeptide (SGP) in egg yolk is composed of six amino acids residues (KVANKT), in which asparagine (N) is modified by sialylated complex N-glycan. SGP is mainly obtained from egg yolk, and currently, the methods for separation and purification of SGP are cumbersome, expensive, and difficult to scale production. In this study, a simple and low-cost method for separation and purification of sialylated glycopeptide from egg yolk was developed based on the hydrophilic interaction chromatography by using medical absorbent cotton as stationary phase. Firstly, 50 egg yolks were treated with phenol to obtain crude sialylated glycopeptide. The obtained glycopeptide was then formulated into a 150 mg/mL solution and loaded onto the prepared cotton hydrophilic chromatographic column, followed by the treatment with 100%, 95%, 85% and 75% acetonitrile aqueous solution to remove the impurity. Finally, the sialyglycopeptide was obtained by eluting with deionized water. By this way, 300 mg SGP was obtained and its purity was identified by high-performance liquid chromatography (HPLC) to be 95%. The glycan composition and glycosylation site of SGP were further validated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS).