Journal of Chromatography B, 809 (2004) 323–329 Lectin affinity capillary electrophoresis in glycoform analysis applying the partial filling technique Maria Bergstr¨ om a,∗ , Mikael Nilsson a , Roland Isaksson a , Ingvar Ryd´ en b , Peter P˚ ahlsson c , Sten Ohlson a a DepartmentofChemistryandBiomedicalSciences,UniversityofKalmar,SE-39182Kalmar,Sweden b DepartmentofClinicalChemistry,KalmarCountyHospital,SE-39185Kalmar,Sweden c DepartmentofBiomedicineandSurgery,DivisionofCellBiology,Link¨ oping University, SE-58185 Link¨ oping, Sweden Received 5 April 2004; received in revised form 25 June 2004; accepted 28 June 2004 Available online 28 July 2004 Abstract The study of protein glycosylation and its significance in biological interactions is a field of growing interest. This work demonstrates a lectin-based separation of protein glycoforms of  1 -acid glycoprotein (AGP or orosomucoid) with capillary electrophoresis. Glycoform analysis was performed with a “partial filling technique” with the lectin Concanavalin A (Con A) as affinity ligand. Con A separated human AGP into two peaks; the first peak included AGP glycoforms without biantennary glycans, and the second peak represented the fraction that had one or more biantennary glycans. The applicability of the method was demonstrated with the analysis of AGP from clinical samples and AGP treated with N-glycosidase F. The AGP separation was also used as a reporter system to estimate the dissociation constant (K D ) between Con A and a competing sugar. © 2004 Elsevier B.V. All rights reserved. Keywords: Partial filling technique;  1 -Acid glycoprotein; Orosomucoid 1. Introduction In recent years, increasing evidence have emerged indi- cating the great importance of carbohydrates in cell differen- tiation, signalling and immune response [1]. Glycosylation is the most common post-translational modification in eukary- otes and it has a great impact on the functional, biophysical and biochemical properties of the glycosylated product. Gly- coproteins can exist in a high number of different glycoforms, differing in number of carbohydrate chains, branching, length and modifications such as sialic acid and fucose. This is also referred to as glycoprotein microheterogeneity. The relative proportions of the different glycoforms of a specific protein are normally constant in a healthy individual. Changes in the glycosylation pattern can be seen in e.g. malignant and ∗ Corresponding author. Fax: +46 480 446262. E-mailaddress: maria.bergstrom@hik.se (M. Bergstr¨ om). inflammatory diseases and have the potential to be used as di- agnostic and prognostic tools [2,3]. The physiological signif- icance of these findings is not yet fully understood and studies of carbohydrate interactions in biological system are of high importance [4]. The analysis of glycoform heterogeneity in the production of recombinant glycoproteins is also of special interest to assure biocompatibility. To determine glycoform distribution it is desirable to analyse the intact glycoprotein rather than the free carbohydrates. The large number of possi- ble glycoforms of an individual protein makes this a difficult task [5]. Capillary electrophoresis (CE) is a promising tool to study both intact glycoproteins, glycopeptides as well as released carbohydrate chains [6]. The heavily glycosylated acute phase protein  1 -acid gly- coprotein (AGP or orosomucoid) is a major constituent of hu- man plasma but its function is still obscure [7]. AGP has five glycosylation sites with N-linked complex-type oligosaccha- ride chains that can be of biantennary, triantennary as well 1570-0232/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2004.06.042