GlycoProfile™ Labeling Kits: Useful Fluorescent Dyes for Enhanced Glycan Analysis

BioFiles 2007, 2.1, 14.

Glycan analysis has become an increasingly critical aspect of glycomics and proteomics, as the role of glycoproteins in cell signaling, cell adhesion, immune response, and disease states is emerging through ongoing research. In contrast to proteins and peptides, glycans do not absorb ultraviolet (UV) light strongly, thereby giving a weak detector signal, even at 214 nm. Furthermore, as glycans with various structures may be present in minute amounts in glycoprotein hydrolysates, their detection by UV absorbance may not be practical.

Most glycoproteins exist as a heterogeneous population of glycoforms or glycosylated variants with a single protein backbone and a heterogeneous population of glycans at each glycosylation site. It has been reported that for some glycoproteins, 100 or more glycoforms exist at each glycosylation site. In view of this heterogeneity and the presence of branched structures, the detailed analysis of glycans can be very complex.

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2-AA and 2-AB Labeling of Glycans by Reductive Animation

Once glycans have been cleaved from the glycoprotein, the glycan pool can be labeled with a fluorescent dye and analyzed by HPLC or MS, or both. This strategy can provide a “glycan profile” or a “glycosylation pattern” that is highly characteristic of the glycoprotein. The methodology can be used to compare glycan profiles of glycoproteins found in normal and diseased states, or to compare different batches of recombinant protein products.

Both the GlycoProfile 2-AA and GlycoProfile 2-AB Labeling Kits contain reagents for labeling glycans at their reducing ends by reductive amination. The fluorophores 2-AA (anthranilic acid) and 2-AB (2-aminobenzamide) provide valuable tools for glycan analysis due to their sensitivity and stability when coupled to glycans. Other commonly used methods, such as radioisotopic labels, antibody labels, and various probes do not display the stability, flexibility, and ease of use observed with 2-AA and 2-AB.

Labeling using 2-AA / 2-AB can be performed on either purified or pooled samples, including a variety of sources, such as N-linked, O-linked, and GPI anchored glycans. For samples containing sialated oligosaccharides, sialic acid loss is negligible.

The coupling reaction proceeds through Schiff’s base formation of an acyclic reducing sugar with the amine moiety of the dye. The bond is subsequently reduced and stabilized during the coupling reaction (see Figure 1).



Figure 1. Acyclic glycan and dye form a Schiff’s base. Subsequent reduction of the imine with sodium cyanoborohydride results in a stable labeled glycan. (A) 2-AA fluorophore (B) 2-AB fluorophore.

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Analysis of 2-AA and 2-AB Labeled Glycans

Once the glycans have been labeled, a variety of methods exist to analyze them. The most common techniques employ fluorescent detection after separation by HPLC or CE. These include separation by ion exchange, normal phase/RP HPLC, and size exclusion chromatography.

The labeled glycans are undetected by UV detection, but significant peaks are seen by fluorescence (see Figures 2 and 3). The different chromatograms are due to the labeling efficiency, sensitivity, and other dye properties. Neither UV nor fluorescent detection was able to detect unlabeled fetuin glycans (data not shown). Labeled glycans can also be detected using mass spectrometry. Mass spectrometry can be performed with either an electrospray ionization (ESI) or matrix assisted laser desorption ionization (MALDI) ion source. Samples containing mixed pools of glycans can often be detected at picomolar concentrations.

Figure 2. HPLC profile of the 2-AA labeled N-linked glycan library obtained from bovine fetuin. The glycans were cleaved from the glycoprotein using the Enzymatic Protein Deglycosylation Kit (Cat. No. E-DEGLY).

Figure 3. HPLC profile of the 2-AB labeled N-linked glycan library obtained from bovine fetuin. The glycans were cleaved from the glycoprotein using the Enzymatic Protein De glycosy lation Kit (Cat. No. E-DEGLY).

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GlycoProfile™ 2-AA and 2-AB Labeling Kits

Each GlycoProfile™ Labeling Kit contains sufficient reagents for labeling up to 36 samples. Two sets of components have been provided; each set is sufficient for labeling up to 18 samples based on a reaction volume of 5 μL. Mixed glycan samples should contain between 100 picomoles to 50 nanomoles of purified glycans. With a single pure glycan, as little as 5 picomoles may be labeled and detected in subsequent HPLC analysis.

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Dextran Ladder

Along with fluorescent labeling of glycans and analysis by normal phase HPLC, an external standard is often used to calibrate the HPLC system. Partially hydrolyzed dextran, consisting of a variable number of monomeric glucose units, may be used as an external standard after fluorescent labeling. This dextran standard has a characteristic ladder profile from monomeric glucose to approximately a 20-mer of glucose oligosaccharide, depending on the chromatographic conditions employed. The elution position of each peak in this ladder is expressed as a glucose unit (gu) and is used to assign gu values to peaks in the released glycan pool.

Dextran Ladder is prepared by partial acid hydrolysis of dextran from Leuconostoc mesenteroides with an average molecular weight of 100-200 kDa. A mixture of α-(1→6) linked glucose oligosaccharides of various lengths is produced. The Dextran Ladder may be fluorescently labeled with Sigma’s GlycoProfile 2-AB or 2-AA Labeling Kits.

The purity and structural integrity of the ladder is assessed by fluorescently labeling an aliquot and subsequent analysis by normal phase HPLC. The separation of the different glucose oligomers on an amide HPLC column is shown (see Figure 4).

Figure 4. Normal phase HPLC chromatograph of Dextran Ladder after fluorescent labeling with 2-AB.1

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Materials

     

References

  1. Guile, G.R., et al., A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem., 240, 210-226 (1996).
  2. Yamashita, K., et al., Analysis of Oligosaccharides by Gel Filtration, Meth. Enzymol., 83, 105-126 (1983).

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