Improved isolation and characterization procedure of sialylglycopeptide from egg yolk powder

Abstract

Sialylglycopeptide (SGP) is a complex bi-antennary N-glycan bearing a short peptide fragment that can be isolated from the yolk of hen eggs. This natural product has gained popularity as a starting material for the semi-synthesis of N-glycans. We have found that current isolation methods provide a glycopeptide contaminated with several related structures, one being a glycopeptide having a hexose directly attached to peptide backbone, most like through the hydroxyl containing side chain of the threonine moiety. Furthermore, current methods employ fresh egg yolks that need to be lyophilized and involve several tedious purification steps. Herein, we report a convenient method for the isolation of gram quantities of homogeneous SGP from commercially available egg yolk powder using solid/liquid extraction and HILIC-HPLC purification.

Top-Down Chemoenzymatic Approach to Synthesizing Diverse High-Mannose N-Glycans and Related Neoglycoproteins for Carbohydrate Microarray Analysis

“…SGP was isolated from hen egg yolk following the previously reported procedure. [36][37][38] The The solution was incubated at RT for 30 min, while monitoring by HPAEC-PAD. Before HPAEC-PAD analysis, the Fmoc was removed by incubating 1 μL of the enzymatic digestion with 1 μL 0.5 NaOH at 60 °C for 5 min.…”

Enzymatic Synthesis of a Fluorogenic Reporter Substrate and a High-Throughput Assay for Fucosyltransferase VIII Provide a Toolkit to Probe and Inhibit Core Fucosylation

“…Given the sensitivity of our developed assay, 1 mg (0.68 µmol) of MU-G0 probe is sufficient for about 3400 individual assays, and therefore efforts towards screening large libraries of compounds will require only milligram-quantities of the probe. This is easily achievable on a lab-scale given established methods to isolate SGP starting material from consumer-grade chicken eggs or egg yolk powder with reported yields of roughly 8 mg per egg [23] or 0.8 mg per g of egg yolk powder [26] (the other starting material, MU-βGlcNAc, being relatively inexpensive from various suppliers).…”

“…We opted for an enzymatic approach to obtain this compound ( Fig. 2) following recent reports detailing both the efficient extraction of sialoglycopeptide (SGP) as a starting material from chicken egg yolk [23][24][25][26] and the use of engineered glycosidases as synthetic tools [27][28][29]. SGP consists of a complex-type disialylated biantennary undecasaccharide-sometimes referred to as the G2S2 glycan-N-linked to the asparagine side-chain of a hexapeptide.…”

“…Sialoglycopeptide (SGP) was extracted and purified from chicken egg yolk powder following previously reported procedures, with slight modifications [26]. Prior to extraction, egg yolk powder was first washed with 95% ethanol.…”

Large scale preparation of high mannose and paucimannose N-glycans from soybean proteins by oxidative release of natural glycans (ORNG)

“…Depending on their source, natural glycans are more likely to be biologically relevant for functional study since they were synthesized by a living cell or organism. Several groups reported isolations of gram-level sialylglycopeptide (SGP) [27,28] and milligram-level asparagine-linked Glc 1 Man 9 GlcNAc 2 [29]. We and others have focused on the use of natural glycans for functional studies using glycan microarray technology, in which hundreds of different, purified glycan structures separated from natural sources can be immobilized onto glass slides for interrogation with glycan binding proteins or microorganisms [30–36].…”