Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin

Abstract

Low-pressure pH gradient ion exchange separation provides a fast, simple and cost-effective method for preparative purification of native and desialylated apo-transferrin. The method enables easy monitoring of the extent of the desialylation reaction and also the efficient separation and purification of protein fractions after desialylation. The N-glycan analysis shows that the modified desialylation protocol successfully reduces the content of the sialylated fractions relative to the native apo-transferrin. In the optimized protocol, the desialylation capacity is increased by 150 %, compared to the original protocol provided by the manufacturer. The molar absorption coefficients in the near-UV region for the native and desialylated apo-transferrin differ by several percent, suggesting a subtle dependence of the glycoprotein absorbance on the variable sialic acid content. The method can easily be modified for other glycoproteins and is particularly appropriate for quick testing of sialic acid content in the protein glycosylation patterns prior to further verification by mass spectrometry.

Keywords: Chromatography; Glycoforms; Molar absorbance; Sialic acid; Transferrin; UV-Vis spectroscopy.

Figure 1

Schematic representation of the microheterogeneity of human transferrin glycoforms.

Figure 2

Low-pressure pH gradient ion exchange separation of native (S+) and desialylated (S-) human transferrin with two distinct signals matching different sialoforms, corresponding to 90 μg of Tf-S and 150 μg of Tf+S (black trace). The pH gradient obtained using pISep buffers is displayed as the red trace.

Figure 3

Structures of various N-glycan residues in the native apo-transferrin, Tf+S (black trace), and desialylated apo-transferrin, Tf-S (blue trace), as determined by UPLC-MS [24]. Schematic N-glycan structures and the corresponding fluorescence signals are indicated by arrows: N-acetylglucosamine (blue), mannose (green), galactose (yellow), fucose (red), sialic acid (pink).

Figure 4

Top: The molar absorption coefficient, ε, of the Tf+S (black) and Tf-S (red) fractions in the near-UV range: ε₂₈₀ (Tf+S) = (84.8 ± 0.2) × 10³ M⁻¹ cm⁻¹ and ε₂₈₀ (Tf-S) = (88.2 ± 0.2) × 10³ M⁻¹ cm⁻¹; Bottom: The difference in molar absorption coefficients, Δε, for the intact and denatured proteins: Tf+S (black trace) and Tf-S (red trace). The values were calculated as Δε = ε_f – ε_u, where ε_f is the molar absorption coefficient of the intact (folded) protein, and ε_u is the molar absorption coefficient of the denatured (folded) protein in 6 M guanidine [25].

Figure 5

Top: The dependence of Tf+S solution absorbance (A₂₈₀) on salt concentration: average values (black) and standard deviations (error bars) for triplicate samples, 0 M < [KCl] < 1.0 M, pH = 7.4; Bottom: The dependence of Tf+S solution absorbance (A₂₈₀) on pH: average values (red) and standard deviations (error bars) for triplicate samples, 4.9 < pH < 7.6, [KCl] = 0.2 M.