Development of a CE-MS method to analyze components of the potential biomarker vascular endothelial growth factor 165

The vascular endothelial growth factor 165 (VEGF(165)) is the predominant form of the complex VEGF-A family. Its angiogenic effect is involved in many physiological and pathological events. For this reason, its roles as a potential biomarker and as a therapeutic drug have been considered. Nevertheless, very little is known about the existence of different forms of VEGF(165) arising from glycosylation and potentially from other PTMs. This aspect is important because different forms may differ in biological activity (therapeutic drug application) and the pattern of the different forms can vary with pathological changes (biomarker application). In this work a CE-MS method to separate up to seven peaks containing, at least, 19 isoforms of intact VEGF(165) is described. Comparison between human VEGF(165) expressed in a glycosylating system, i.e. insect cells, and in a non-glycosylating system, i.e. E. coli cells, has been carried out. The method developed provides structural information (mass fingerprint) about the different forms of VEGF(165) and after the deconvolution and the analysis of the MS spectra, PTMs pattern of VEGF(165) including glycosylation. and loss of amino acids at the N- and C-terminus was identified. Glycans involved in PTMs promoting different glycoforms observed in the CE-MS fingerprint were confirmed by MALDI-MS after deglycosylation with peptide N-glycosidase F. This approach is a starting point to study the role of VEGF(165) as a potential biomarker and to perform quality control of the drug during manufacturing. To our knowledge this is the first time that a CE-MS method for the analysis of VEGF(165) has been developed.