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. 2015 Aug 4;87(15):7754-62.
doi: 10.1021/acs.analchem.5b01340. Epub 2015 Jul 14.

A Method for In-Depth Structural Annotation of Human Serum Glycans That Yields Biological Variations

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Free PMC article

A Method for In-Depth Structural Annotation of Human Serum Glycans That Yields Biological Variations

Ting Song et al. Anal Chem. .
Free PMC article

Abstract

Glycosylation is an important post-translational modification of proteins present in the vast majority of human proteins. For this reason, they are potentially new sources of biomarkers and active targets of therapeutics and vaccines. However, the absence of a biosynthetic template as in the genome and the general complexity of the structures have limited the development of methods for comprehensive structural analysis. Even now, the exact structures of many abundant N-glycans in serum are not known. Structural elucidation of oligosaccharides remains difficult and time-consuming. Here, we introduce a means of rapidly identifying released N-glycan structures using their accurate masses and retention times based on a glycan library. This serum glycan library, significantly expanded from a previous one covering glycans released from a handful of serum glycoproteins, has more than 170 complete and partial structures and constructed instead from whole serum. The method employs primarily nanoflow liquid chromatography and accurate mass spectrometry. The method allows us to readily profile N-glycans in biological fluids with deep structural analysis. This approach is used to determine the relative abundances and variations in the N-glycans from several individuals providing detailed variations in the abundances of the important N-glycans in blood.

Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Extracted compound chromatograms (ECCs) of a monosialylated diantennary species (putative structure inset) before and after exoglycosidase digestion yield the linkage and position of the sialic acid. (a) Chromatogram of N5401 from an HPLC fraction before enzyme digestion. The neutral mass of 1933.71 Da was used to produce the ECC. (b) Chromatogram of the N-glycan after digestion with α(2–3) sialidase for 1 h at 37 °C. The neutral mass of 1642.61 Da is used to produce the ECC. (c) Chromatogram of the N-glycan residue after digestion for 24 h with a cocktail of exoglycosidases, including β (1–4) galactosidase, β N-acetylglucosaminidase, and α(1–2,3) mannosidase, at 37 °C of this fraction. The neutral mass of 1406.53 Da is used to produce the ECC.
Figure 2
Figure 2
Tandem mass spectra of the four isomers of N5402 (symbolic structures inset and the complete structures as elucidated by exoglycosidases).
Figure 3
Figure 3
(a) ECC of mass 2224.80 Da from the library. (b) ECC of mass 2224.80 Da from the commercial serum N-glycans run together with study samples. (c) ECC of mass 2224.80 Da from an individual serum of the study sample.
Figure 4
Figure 4
Total compound chromatogram of the reference library with peaks annotated for structures. Listed are the 100 most abundant components. Less abundant components are not labeled for the sake of clarity. The annotation numbers correspond to structures provided in Table S1 of the Supporting Information.
Figure 5
Figure 5
Variations in relative abundances of the N-glycan subclasses for the nine individual human serum samples.
Figure 6
Figure 6
Human serum “glycan wheel” based on the relative abundances averaged for the nine individual sera.

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