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Review
. 2012 Dec;6(11-12):596-608.
doi: 10.1002/prca.201200045.

Solid-phase glycan isolation for glycomics analysis

Shuang Yang  1 , Hui Zhang
Affiliations
Review

Solid-phase glycan isolation for glycomics analysis

Shuang Yang et al. Proteomics Clin Appl. 2012 Dec.

Abstract

Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. Methods relied on MS require isolation of glycans from negligible salts and other contaminant ions since salts and ions may interfere with the glycans, resulting in poor glycan ionization. To accomplish those objectives, glycan isolation and clean-up methods including SPE, liquid-phase extraction, chromatography, and electrophoresis have been developed. Traditionally, glycans are isolated from proteins or peptides using a combination of hydrophobic and hydrophilic columns: proteins and peptides remain on hydrophobic absorbent while glycans, salts, and other hydrophilic reagents are collected as flowthrough. The glycans in the flowthrough are then purified through graphite-activated carbon column by hydrophilic interaction LC. Yet, the drawback in these affinity-based approaches is nonspecific binding. As a result, chemical methods by hydrazide or oxime have been developed for solid-phase isolation of glycans with high specificity and yield. Combined with high-resolution MS, specific glycan isolation techniques provide tremendous potentials as useful tools for glycomics analysis.

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Conflict of interest statement

The authors have declared no conflict of interest.

Figures

Figure 1
Figure 1
Schematic illustration of protein glycosylation in malignant cells. The glycosylated proteins are released from malignant cells carrying disease-related carbohydrate epitopes into the interstitial space, whether glycoproteins further reach the circulation. Reprint and permission from [11].
Figure 2
Figure 2
Schematic diagram of glycan capture and release on solid-phase via dynamic covalent chemistry. Glycans are catalyzed by addition of aniline to formation of Schiff-based intermediate and further reacted to hydrazide or amine. The hydra-zone bond is stable at pH 7 or mild base condition, but can be easily hydrolyzed in acid condition e.g., 10% formic acid solution. Reprinted with permission from [28].
Figure 3
Figure 3
Schematic illustration of glycan capture procedure by reversible hydrazone solid-phase glycan extraction (rHSPE). Glycoproteins are first trypsin digested; glycans are then released by either PNGase F for N-linked glycans or β-elimination for O-linked glycans; glycans are conjugated to hydrazide beads; nonglycan molecules are removed by washing; conjugated glycans are finally hydrolyzed from solid phase either by acid hydrolysis or competition reaction by formaldehyde solution. Reprinted with permission from [28].
Figure 4
Figure 4
Glycan isolation from human serum with addition of standard glycans and peptides by hydrazide beads. The bottom spectrum was the sample mixture without glycan isolation. The top spectrum was isolated glycans from hydrazide beads after conjugation, washing, and hydrolysis. Reprinted with permission from [28].
Figure 5
Figure 5
Solid-phase glycoblotting and transoximization for glycomics and glycoproteomics analyses. Digested glycopeptides are oxidized on antennary galactose; C-6 oxidized glycopeptides are conjugated with oxime to immobilize glycopeptides; glycopeptides are labeled with tag by transoximization reaction, or glycopeptides are released from solid-phase by PNGase F treatment. Reprinted with permission from [51].
Figure 6
Figure 6
Methods for solid-phase capture and release of underivatized glycans. (i) Hydrazide chemistry for reducing end capture release. (A) pH 3–6 in methanol-acetic acid, microwave, 20 min. (B) 10% formic acid, or 200 mM formaldehyde at room temperature; (ii) Hydroxylamine chemistry for reducing end capture release. (C) DMF-water (1:1), pH 4.8, 40°C, 24 h. (D) 4N HCl hydrolysis; (iii) cysteinamide chemistry for reducing end capture release. (E) ACN-water (1:2), pH 4.0, room temperature, 48 h. (F) KCL in DI, 50°C, pH <6, (acetate and acetic acid); (iv) disulfide chemistry for reducing end. (G) DMSO-acetic acid (7:3), 50 mM sodium cyanoborohydride, 65°C, 3 h. (H) Disulfide bond can be cleaved by 16 mM DTT, 60°C, 1 h; (v) Chemoselective reductive alkylation chemistry for amine glycans. (I) Sodium cyanoborohydride in DMF-acetic acid (99:1). (J) Glycan is released in acetic anhydride-pyridine, TFA-water (19:1) [72]. Reprinted with permission from [57].
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