The utility of nonspecific proteases in the characterization of glycoproteins by high-resolution time-of-flight mass spectrometry

Degradation of glycoproteins with the nonspecific protease pronase was examined by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS). High mass resolution (in excess of 10000 at FWHM) and mass accuracy on monoisotopic species (better than 10 ppm) obtained with the combination of delayed extraction and the reflector mode of analysis enabled the successful interpretation of very complex mixtures resulting from extensive hydrolysis. After 48 h of degradation of glycoproteins, the glycopeptides were well separated from small peptides based on their molecular weight. Accurate monoisotopic masses of the glycopeptides permitted the determination of the glycan composition and the (nonspecific) peptide segment the glycans were attached to. Ribonuclease B and ovalbumin were used to demonstrate feasibility of the method. Unless the peptide sequence bf the glycopeptide contains a basic residue, the negative-ion mode is preferred over the positive-ion mode. The absence of alkali adducts reduce the complexity of the mass spectra and the relative sensitivities of the glycopeptides were found better in the negative-ion mode. A highly complex sample, alpha-acid glycoprotein was also analyzed. Multiply sialylated glycopeptides had to be run in the linear mode where the metastable loss of sialic acid residues did not interfere with the detection of other components. From the linear mode mass spectrum, five glycosylation sites were identified along with the more abundant glycoforms. Comparison with the literature indicated that several minor components were undetected. The presented approach also permitted the determination of the sialylation pattern of the complex type glycans. (C) 1997 Elsevier Science B.V.