MASS-SPECTROMETRIC DETERMINATION OF THE SITES OF O-GLYCAN ATTACHMENT WITH LOW PICOMOLAR SENSITIVITY

A sensitive protocol for unambiguously and positively identifying O-gl ycosylation sites in glycopeptides is described, based on beta-elimina tion of the glycan chain(s) using NH4OH. On glycan elimination, NH3 is incorporated into the amino acid residue(s) to which the glycan(s) ha d been attached, to yield a modified amino acid residue having a disti nct mass. Electrospray ionization collision-induced dissociation tande m mass spectrometry allows the released, modified peptide to be sequen ced and the site(s) of the modified amino acid residue(s) to be identi fied. The protocol has been optimized using a series of structurally r elated O-glycopeptides, and standard conditions are recommended for ha ndling unknowns, We demonstrate that site determination can be achieve d using as little as 1 pmol of starting material. (C) 1998 Academic Pr ess.