Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS. The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation. The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides. The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program. The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes for beyond previously published data and sometimes differs from them. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala).
Zeng
C
C
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139-4307, USA.
Biemann
K
K
D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000032:analysis* / Q000737:chemistry; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D016415:Sequence Alignment; D043324:beta-Fructofuranosidase
D005656:Fungal Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase