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

Abstract

The vascular endothelial growth factor 165 (VEGF165) 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 VEGF165 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 VEGF165 is described. Comparison between human VEGF165 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 VEGF165 and after the deconvolution and the analysis of the MS spectra, PTMs pattern of VEGF165 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 VEGF165 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 VEGF165 has been developed.

Capillary electrophoresis–mass spectrometry of intact basic proteins using Polybrene–dextran sulfate–Polybrene-coated capillaries: System optimization and performance

“…Moreover, the sheath liquid composition and flow rate can be specifically optimized to enhance MS signal intensities [10][11][12]. Sheath liquid CE-MS of, for example, glycoproteins [13], biopharmaceuticals [14], metalloproteins [15] and dietary proteins [16] has been described.…”

Capillary electrophoresis‐mass spectrometry for analysis of complex samples

“…The separation and re‐equilibration time is shorter, and the cost of capillaries is much lower than that of HILIC columns. Many people reported glycan analysis based on neutrally coated capillaries while research employing bare fused‐silica capillary also yielded satisfactory results .…”

On‐line CE/ESI/MS interfacing: Recent developments and applications in proteomics

“…A majority of CE/MS separations of intact proteins is focused on the analysis of biotechnological and biopharmaceutical products Detailed characterization of recombinant protein biopharmaceuticals represents a key step during the drug development or quality control processes. Another frequent application of CE/MS is searching for potential biomarkers associated with various diseases or pathological conditions or monitoring of blood doping such as uptake of erythropoietin or artificial oxygen carriers . In all of these fields, simple and fast analytical methods, such as CE, are required in order to minimize sample complexity and to obtain clear mass spectra.…”

“…Coupling of high‐resolution separation methods to high‐resolution MS has the potential to be a powerful tool for identification of intact proteins and individual protein isoforms by exact mass measurements. For example, a potential biomarker and a therapeutic drug, vascular endothelial growth factor 165 (VEGF 165 ), has been studied by CE in combination with high resolution TOF/MS . Up to 7 peaks containing at least 19 isoforms of intact recombinant human VEGF 165 were separated using a cationic poly‐LA 313‐coated capillary and a BGE consisting of 60 mmol/L formic acid (Fig.…”

“…Extracted ion electrophoreograms (A) and MS spectrum of time window 38.8–39.1 min (B). Reproduced with permission from .…”