The Characterization And Post-translational Modification Of EDGP From Carrot (Daucus Carota.L.)

Extracellular dermal glycoprotein (EDGP), with apparent molecular weight of 57000 on SDS-PAGE, is constituted of two pi values of 8.8 and 9.5 with similar amino acid composition. EDGP was purified by chromatography and chromatofocussing from the ammonium sulfide-precipitation of carrot cell (Daucus carola L.) suspension culture medium with 2,4-dichlorophenoxyacetic acid. The purified protein was identified as EDGP by matrix-assisted laser desorption/ ionization - time of flight mass spectrometry (MALDI-TOF-MS) and MS-Fit. a peptide mass fingerprinting tool on the Internet. By electrospray mass spectrometry(ESI-MS), the accurate molecular weight of EDGP was determined as the following: 48119 Da, 48282 Da, 48479 Da, and 48705 Da. The amino acid .sequence of EDGP deduced from the nucleotides sequence was further confirmed by MALDI-TOF-MS. In MALDI-TOF mass spectra of EDGP tryptic digests, the following peptides sequence were observed: Tl. T2, T3, T4, T5, T8, T9. T10. Til. T12. T15, T19. T20. T21. T23, T25 and T26. In the amino acid sequence of EDGP. the first 20 residues belong to signal peptide. and the N-terminal glutamine was modified by pyroglutamic acid. There are 11 potential phosphorylation sites in EDGP. seven of them. Tl. T5. Til. T15, T19. T23, and T25. has been found not to be phosphorylated.It was confirmed that there is no free cysteine in EDGP by fluorescence probe method. Determination of peptide mapping with and without reducing agent followed by MALDI-TOF-MS indicated that the cysteine 70 is linked with the cysteine 158 by disulfide bond.