Traditional N-glycan characterization methods
Various methods are used to characterize N-glycans. Figure 1 summarizes those most commonly used. Most require multiple steps
and a day or more to complete. All typically follow the same basic process as follows:
- Release of glycans from glycoproteins
- Separation of glycans from the deglycosylated protein
- Analysis (separation, detection, identification, and quantification) of glycans with or without derivatization.
Enzymatic digestion with PNGase F ((peptide-N4-(N-acetyl-β-D-glucosaminy-I)) asparagine amidase F; EC 220.127.116.11) is commonly
used to release glycans from glycoproteins. PNGase F is an amidase that cleaves the amide bond between the glycosylation-site
asparagine and the innermost N-acetyl-β-D-glucosamine residue of N-glycans. It has a wide specificity, releasing all major
classes of N-glycans found on mammalian glycoproteins. As shown in Figure 1, this method of deglycosylation involves long
incubation times of 3 to 20 h, a significant component of the overall analysis time.
After releasing the glycans, the next step is to separate and recover them from the deglycosylated protein. This step is commonly
done using reverse-phase or graphitized carbon-based solid-phase extraction, ultrafiltration, and precipitation. Centrifugal
evaporation is typically needed to concentrate and dry the purified glycans. Depending on the method used, this step can take
several hours to complete (see Figure 1).
Various methods are used to separate and detect glycans. Although high pH anion-exchange chromatography (HPAEC), with pulsed
amperometric detection (PAD), has been popular because it does not require derivatization (4), it is being replaced by newer
methods such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) with fluorescence detection.
To improve the separation and enhance detection sensitivity, these methods involve derivatizing free glycans with fluorophore
(via reductive amination.) After separation, the glycans' retention time or migration time is measured, and peaks are correlated
with standards. Relative quantitation is performed based on the fluorescence of individual peaks (5). For CE analysis, glycans
are usually labeled with fluorescent tag APTS (8-aminopyrene-1,3,6-trisulfonic acid.) (6). For HPLC analysis, glycans are
typically labeled with a fluorescent label such as 2-aminopyridine (2-AP), anthranilic acid (2-AA), or 2-aminobenzamide (2-AB),
and separation is performed using either hydrophilic interaction chromatography (HILIC) or reverse-phase chromatography. Depending
on the method used, labeling requires about 1–3 h to complete. Postlabeling cleanup further increases the time requirements
needed to complete the analysis.