Capillary Isoelectric Focusing–Electrospray Ionization Mass Spectrometry for Transferrin Glycoforms Analysis

On-line capillary isoelectric focusing (CIEF)–electrospray ionization mass spectrometry (ESIMS) as a two-dimensional separation system is employed for high-resolution analysis of bovine serum apotransferrin glycoforms. On the basis of their differences in isoelectric point (pI), the di-, tri-, and tetrasialotransferrins are separated and resolved in CIEF. The focused protein zones of di-, tri-, and tetrasialotransferrins are eluted by combining gravity with cathodic mobilization. At the end of CIEF capillary, the mobilized transferrin zones are analyzed by mass spectrometry coupled on-line to an electrospray interface with a coaxial sheath flow configuration. Additional transferrin variants within each of di-, tri-, and tetrasialotransferrins, differing in their molecular weights, are easily distinguished by ESIMS. In combination with sialidase digestion, the distribution of pIand molecular weight of asialo-, mono-, di-, tri-, and tetrasialotransferrin variants was obtained from the CIEF–ESIMS measurements. In addition to the differences in the number of sialic acid, the microheterogeneity of bovine serum apotransferrin glycans might be complicated by the partial fucosylated structure and the α-Gal (1–3)-β-Gal on the α-Man-(1–6) antenna.