Abstract
Sporamin is a vacuolar storage protein of sweet potato. This protein, when expressed in tobacco BY-2 cells, is hydroxylated at Pro36 residue and further modified by O-glycosylation. In the present study, we analyzed the primary structure of the glycosyl residue motif using MALDI-TOF MS.
Sporamin was first denatured in SDS solution, and subsequently digested by 0.5 pmol of Trypsin or Endoproteinase Asp-N, respectively. The digested sporamin was directly spotted on sample plate. Both secretory mutant sporamin and vacuolar wild-type sporamin were hydroxylated and glycosylated in contrary to recombinant sporamin prepared from E. coli. Both glycosylated sporamins contained degree of polymerization up to 9 hexoses with one to three pentoses, as judged from MALDI-TOF MS results.We are currently analyzing the glycosylation pattern of several mutant sporamins that have replaced single amino acid residues around Pro36 with alternative amino acids.
