2 research outputs found
CIEF and MALDI-TOF-MS methods for analyzing forms of the glycoprotein VEGF(165)
The vascular endothelial growth factor (VEGF) is involved in different sicknesses
(cardiovascular diseases, cancer, and other). Out of the many components of the VEGF
family, the A splice variant with 165 amino acids (VEGF165) is the main component.
In spite of the potential as biomarker that this protein has, information about its
physico-chemical characteristics is scarce. In this study CIEF and MALDI-TOF-MS
methods for intact recombinant human VEGF165 are developed and applied to
analyze this glycoprotein expressed in glycosylating (Sf 21 insect cells) and non-glyco-
sylating (Escherichia coli) systems. Different parameters influencing the CIEF separation
were studied. The developed CIEF method allowed for the separation of up to seven
peaks in the VEGF165 expressed in insect cells and up to three in VEGF165 expressed in
E. coli. The use of the presented method permits the estimation of the apparent pI of the
different forms of VEGF165 expressed in insect cells to be in a range of 6.8–8.2. The three
peaks with intermediate pI values are observed in the protein expressed in both systems,
insect cells and E. coli. The MALDI-TOF-MS method enabled to a rapid partial char-
acterization of VEGF165 based on its MS fingerprint. MALDI-MS analysis of VEGF165
expressed in insect cells shows the presence of, at least, four forms or groups of forms of
VEGF165 as a result of the different PTMs of the protein. According to the MALDI-MS
analysis, VEGF165 expressed in E. coli was produced as a very homogeneous protein,
although the results suggest the existence of some PTMs in the protein. The patterns of
VEGF165 of both origins obtained by CIEF and MALDI-MS indicate the possibility of
using these analytical methods to compare samples from people with different patho-
physiological conditions. This work is thus a starting point to make possible the study of
the role of the various forms of VEGF165 as biomarkers. Finally, to the best of our
knowledge, this is the first time that intact VEGF165 has been analyzed by CIEF and
MALDI-TOF-MS.Peer reviewe