Improved strategy for large scale isolation of sialylglycopeptide (SGP) from egg yolk powder

Graphical abstract


Specifications Table
Subject Area: Agricultural and Biological Sciences More specific subject area: Natural product isolation Analytical glycobiology Method name: Improved strategy for large scale isolation and purification of sialylglycopeptide from egg yolk powder Name and reference of original method: This method was significantly modified for isolating the glycosylated hexapeptides as described by Seko

Method details
The ability to produce defined and well-characterised compounds by synthetic approaches has always been a major driving force in science. The establishment of synthetic routes allowing the production of defined DNA or peptide sequences in high yields and purity has been crucial for the development of biosciences and our current understanding of cellular functions [1,2]. However, to date the synthetic capacities to easily produce a large variety and quantity of synthetic glycopeptides is still lagging behind classical peptide or DNA synthesis strategies, also because of the large structure complexity associated with glycans.
Despite tremendous advances in carbohydrate chemistry [3][4][5] the production of large oligosaccharides with diverse building blocks still requires substantial time and material resources. In particular, introduction of certain biologically important glyco-features such as α-fucose, α-sialyl or β-mannose linkages between the monosaccharide building blocks are challenging and represent major limiting steps in the production of sufficient quantities of larger oligosaccharides such as N-glycans. Despite these challenges, total complete synthesis of glycopeptide bearing fucosylated biantennary, disialylated N-glycans was in principle accomplished by the Danishefsky research group [6], though with considerable efforts that yet make it unfeasible for routine production.
One way of circumventing these obstacles is to make use of nature's glycosylation potential by isolating the compounds of interest from natural resources such as egg yolk [7][8][9][10][11]. These compounds can easily be purified and subsequently transformed into protected building blocks, which then can be used for glycoconjugate synthesis [7][8][9][10][11]. This approach is significantly more cost effective, quicker and allows the production of g-quantities within just 2-3 weeks. Thus, having a methodology in hand that allows easy production of the necessary precursor for chemo-enzymatic synthesis of glycoconjugates will significantly contribute towards better understanding of the biological role of glycoconjugates and their analysis [12][13][14][15][16]. Here we present the significant improvement from earlier strategies [7,[17][18][19][20] that reduces the time to isolate milligram quantities of glycosylated amino acid precursors from egg yolk by 80%. The here described simple and environmentally friendly isolation strategy provides the crucial precursor building blocks necessary for step-wise chemo-enzymatic synthesis glycoconjugates required for functional and analytical glycobiology.

Procedure
Seko and co-workers first reported the isolation of a SGP from fresh chicken egg yolks in milligram scale quantities [17]. However, this approach came with the major drawback that large amounts of phenol were required representing significant downstream processing issues as phenol waste represents a considerable environmental hazard. Here we propose an alternative strategy to isolate a SGP and also show that similar yields could be achieved from commercially available chicken egg yolk powder using a chloroform/methanol protocol [21] that concomitantly precipitated proteins and removed lipids into different fractions and also allowed recycling of the used organic solvents.
Sialylglycopeptide extraction 1) Resuspend 250 g of egg yolk powder in 750 ml of MilliQ (MQ) water (1: 3 = W/V ratio) and stir the suspension for 2 h at room temperature.
Note: resuspension step can also be carried out overnight if required at room temperature 1) Add, 500 ml of Methanol (2 vol) to this mixture and stir for another 1 h at room temperature.
2) Separate the suspension into fractions of 40 ml using 50 ml falcon tubes.
3) Centrifuge at 3000 x g at 4 C for 5 min 4) Add 10 ml chloroform to the egg yolk-Methanol mixture and mix well 5) Centrifuge at 3000 x g at 4 C for 10 min 6) Carefully, decant the aqueous phase containing the desired product and concentrate the pooled fractions under reduced pressure using a rotovap.

Purification
The obtained crude product was volume reduced using rotovap and the concentrated solution was subjected to gel filtration on Sephadex G50 (fine) (25 X 935 mm) and eluted with 100 mM Ammonium

Method validation
This novel isolation workflow comes with an additional advantage that just a single chromatographic step after chloroform/methanol precipitation is sufficient to yield the glycosylated hexapeptide and the isolated product was verified by ESI-MS/MS (Figs. 1 and 2). The ratio used for extraction of the glycosylated hexapeptide from egg yolk enabled a quick and easy isolation of approx. 200 mg glycopeptide from 250 egg yolk (Table 1). One of the biggest advantages of the procedure is that the chloroform layer containing the lipids can easily be reused after a simple distillation, thus tremendously reducing the amounts of organic solvents required for glycopeptide purification and reducing organic waste in general.  [22].