TY - JOUR
T1 - Structural elucidation of O-linked glycopeptides by high energy collision-induced dissociation
AU - Medzihradszky, K. F.
AU - Gillece-Castro, B. L.
AU - Townsend, R. R.
AU - Burlingame, A. L.
AU - Hardy, M. R.
N1 - Funding Information:
The authors would like to thank F. C. Walls for his technical assistance and F. R. Masiarz for the Edman data. We are indebted to L. Otvos, Jr. (Wistar Institute, Philadelphia) for supplying the synthetic glycopeptides. This work was supported by grants from the National Center for Research Resources, National Institutes of Health (Grant RRO1614), and the National Science Foundation (Grant 8700766).
PY - 1996/4
Y1 - 1996/4
N2 - O-linked glycopeptides that bear a GalNAc core with and without the presence ot sialic acid have been analyzed by high energy collision-induced dissociation (CID). We show that the CID spectra from the glycosylated precursor ions contain sufficient information to identify the peptide sequence and to determine the glycosylated site(s). Asialo O-linked glycopeptides, previously prepared from a tryptic digest of bovine fetuin were studied. One of the glycopeptides contained only a single Hex (hexose)-HexNAc (N-acetylhexosamine) substitution at Thr262, whereas the other exhibited Hex-HexNAc moieties at both Thr262 and Ser264. In addition, sialo and asialo fetuin glycopeptides from a pronase digest were derivatized with t-butoxycarbonyl-tyrosine, and characterized by high energy CID analysis. The presence of a Gal β(1,3)GalNAc core structure at Ser264 was confirmed by using the substrate specificity of endo-α-N-acetylgalactosaminidase. These studies revealed the presence ot a β-galactosidase specific for β(1,4) linkages in the endo-α-N-acetylgalactosaminidase preparation employed. Finally, the relative stability of N- and O-glycosyl bonds to high energy CID is addressed based upon comparison of the behavior of a synthetic N-linked glycopeptide with analogous O-linked structures.
AB - O-linked glycopeptides that bear a GalNAc core with and without the presence ot sialic acid have been analyzed by high energy collision-induced dissociation (CID). We show that the CID spectra from the glycosylated precursor ions contain sufficient information to identify the peptide sequence and to determine the glycosylated site(s). Asialo O-linked glycopeptides, previously prepared from a tryptic digest of bovine fetuin were studied. One of the glycopeptides contained only a single Hex (hexose)-HexNAc (N-acetylhexosamine) substitution at Thr262, whereas the other exhibited Hex-HexNAc moieties at both Thr262 and Ser264. In addition, sialo and asialo fetuin glycopeptides from a pronase digest were derivatized with t-butoxycarbonyl-tyrosine, and characterized by high energy CID analysis. The presence of a Gal β(1,3)GalNAc core structure at Ser264 was confirmed by using the substrate specificity of endo-α-N-acetylgalactosaminidase. These studies revealed the presence ot a β-galactosidase specific for β(1,4) linkages in the endo-α-N-acetylgalactosaminidase preparation employed. Finally, the relative stability of N- and O-glycosyl bonds to high energy CID is addressed based upon comparison of the behavior of a synthetic N-linked glycopeptide with analogous O-linked structures.
UR - http://www.scopus.com/inward/record.url?scp=0029866539&partnerID=8YFLogxK
U2 - 10.1016/1044-0305(95)00682-6
DO - 10.1016/1044-0305(95)00682-6
M3 - Article
C2 - 24203358
AN - SCOPUS:0029866539
SN - 1044-0305
VL - 7
SP - 319
EP - 328
JO - Journal of the American Society for Mass Spectrometry
JF - Journal of the American Society for Mass Spectrometry
IS - 4
ER -