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CIEF and MALDI‐TOF‐MS methods for analyzing forms of the glycoprotein VEGF 165
Author(s) -
Ongay Sara,
Puerta Angel,
DíezMasa Jose Carlos,
Bergquist Jonas,
de Frutos Mercedes
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800592
Subject(s) - glycoprotein , vegf receptors , vascular endothelial growth factor , escherichia coli , recombinant dna , chemistry , microbiology and biotechnology , biology , chromatography , biochemistry , gene , cancer research
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 (VEGF 165 ) 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 VEGF 165 are developed and applied to analyze this glycoprotein expressed in glycosylating ( Sf 21 insect cells) and non‐glycosylating ( 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 VEGF 165 expressed in insect cells and up to three in VEGF 165 expressed in E. coli . The use of the presented method permits the estimation of the apparent p I of the different forms of VEGF 165 expressed in insect cells to be in a range of 6.8–8.2. The three peaks with intermediate p I 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 characterization of VEGF 165 based on its MS fingerprint. MALDI‐MS analysis of VEGF 165 expressed in insect cells shows the presence of, at least, four forms or groups of forms of VEGF 165 as a result of the different PTMs of the protein. According to the MALDI‐MS analysis, VEGF 165 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 VEGF 165 of both origins obtained by CIEF and MALDI‐MS indicate the possibility of using these analytical methods to compare samples from people with different pathophysiological conditions. This work is thus a starting point to make possible the study of the role of the various forms of VEGF 165 as biomarkers. Finally, to the best of our knowledge, this is the first time that intact VEGF 165 has been analyzed by CIEF and MALDI‐TOF‐MS.