Determination of the C-terminus of p110 sEGFR
EMSL Project ID
3100
Abstract
A 110 kDa, soluble fragment of the EGFR protein (p110 sEGFR) has been identified as a circulating marker of cancer (Maihle and coworkers, 1998, 1999). The p110 sEGFR is released into the serum as the results of the action of a specific protease that cleaves the full-length, transmembrane EGFR protein and thereby releases the soluble extracellular portion of the molecule. Knowledge of the C-terminal sequence of the p110 sEGFR would identify the proteolytic cleavage site and facilitate identifying the protease responsible for the cleavage process. We propose testing technology currently being utilized by Dave Camp to identify the C-terminus. This approach uses methanolic HCl to label carboxylic acid groups with a methyl ester, and therefore should selectively label the C-terminus carboxyl group of the intact protein. The availability of duteriated reagents allows for the isotopic labeling of the samples, providing greater confidence in peptide identification. Since this procedure has only been tested with tryptic peptides, initial studies will be conducted with whole albumin to ensure that the process works with intact protein. Once work with whole proteins has been validated, purified full-length EGFR and p110 sEGFR (provided by Nita Maihle, Mayo Clinic) will be analyzed. Tryptic digests of the labeled protein will be analyzed by LCQ MS and the tandem MS spectra analyzed using SEQUEST and a database containing only the EGFR protein sequence.
Project Details
Project type
Exploratory Research
Start Date
2003-02-03
End Date
2004-04-02
Status
Closed
Released Data Link
Team
Principal Investigator