An Efficient and Economical N-Glycome Sample Preparation Using Acetone Precipitation

Abstract

Due to the critical role of the glycome in organisms and its close connections with various diseases, much time and effort have been dedicated to glycomics-related studies in the past decade. To achieve accurate and reliable identification and quantification of glycans extracted from biological samples, several analysis methods have been well-developed. One commonly used methodology for the sample preparation of N-glycomics usually involves enzymatic cleavage by PNGase F, followed by sample purification using C18 cartridges to remove proteins. PNGase F and C18 cartridges are very efficient both for cleaving N-glycans and for protein removal. However, this method is most suitable for a limited quantity of samples. In this study, we developed a sample preparation method focusing on N-glycome extraction and purification from large-scale biological samples using acetone precipitation. The N-glycan yield was first tested on standard glycoprotein samples, bovine fetuin and complex biological samples, and human serum. Compared to C18 cartridges, most of the sialylated N-glycans from human serum were detected with higher abundance after acetone precipitation. However, C18 showed a slightly higher efficiency for protein removal. Using the unfiltered human serum as the baseline, around 97.7% of the proteins were removed by acetone precipitation, while more than 99.9% of the proteins were removed by C18 cartridges. Lastly, the acetone precipitation was applied to N-glycome extraction from egg yolks to demonstrate large-scale glycomics sample preparation.

Keywords: LC-MS/MS; acetone precipitation; glycan; oxidative release; permethylation.

Figure 1

(A) Extracted Ion Chromatograms (EICs) of permethylated N-glycans derived from bovine fetuin treated by acetone precipitation (red trace) and C18 cartridges (black trace). (B) Quantification of N-glycans based on the area under the peak (n = 3, *: p < 0.05). (C) Total abundance of N-glycans detected in the samples prepared by acetone precipitation and C18 cartridges. Symbols: , N-acetylglucosamine (GlcNAc); , Galactose (Gal); , Fucose (Fuc); , Mannose (Man); , N-acetylneuraminic acid (NeuAc/Sialic Acid).

Figure 2

TICs of N-glycomics samples derived from 1 μL of human serum after purified by using acetone (red trace) and C18 cartridges (black trace). Symbols of monosaccharides are the same as shown in Figure 1.

Figure 3

Comparisons of 61 N-glycan abundances from 1 μL of human serum treated by acetone precipitation and C18 cartridges. (n = 3) (A) The N-glycans showing higher average abundances purified by acetone. (B) The N-glycans showing higher abundances purified by C18 cartridges. (C) Abundances of total N-glycans. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Symbols of monosaccharides are the same as shown in Figure 1.

Figure 4

Quantification results of N-glycans extracted from egg yolks using oxidative release followed by acetone precipitation. (A) Absolute abundance of 42 identified N-glycans. (B) Distribution of five types of N-glycans. Symbols of monosaccharides are the same as shown in Figure 1.