281614 Deglycosylation of Receptor Tyrosine Kinases to Improve Resolution On 2D Gels

Tuesday, October 30, 2012: 9:00 AM
Fayette (Westin )
Nancy Kendrick1, Matt Hoelter1, Jon Johansen1 and Mary Ann Gawinowicz2, (1)Kendrick Labs Inc, Madison, WI, (2)Protein Core Facility, Columbia University, New York, NY

The human genome is known to encode 58 receptor tyrosine kinase proteins (RTK) that regulate cell division and differentiation in both normal tissue and malignant tumors. Binding of a serum ligand to these high molecular weight transmembrane proteins induces protein dimerization followed by phosphorylation of tyrosine residues on the intracellular tails. RTK tyrosine phosphorylation triggers cascades of reactions that result in cell division; it is known to drives cancer growth.   We have previously shown that tyrosine phosphorylation of RTKs can be detected using carrier ampholine 2-dimensional electrophoresis (2DE) in conjunction with Western blotting. However, RTK proteins are heavily glycosylated and appear as large, diffuse spots on 2D gels  because of microheterogeneity of the sugar chains. The glycosylation makes it difficult to identify RTK proteins cut from 2D gels using mass spectrometry, and also difficult to distinguish between  RTKs using Western blotting. Removal of the sugar chains should collapse the 2D pattern and remedy both these problems. 

Deglycosylation enzymes are now commercially available. As a test, recombinant PNGase F was purchased and used to remove N-linked sugar chains from recombinant Vascular Endothelial Growth Factor Receptor (VEGF, C-terminal fragment) from ProQinase that had been glycosylated by the host sf9 insect cells. The deglycosylated pattern was compared to the original usinge 2DE. Additional treatment with O-glycosidase to remove O-linked sugar chains was performed as well as treatment with sialidase to remove terminal sialic acids. The treated and untreated samples were run on 2D gels and the patterns compared for differences. VEGF protein spot was cut from the condensed regions and analyzed using LC/MS/MS to determine sensitivity. Once conditions were optimized, human lung tumor homogenates were deglycosylated and analyzed by 2DE with phosphotyrosine Western blotting followed by protein identification using LC/MS/MS.

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