Nanospray-ESI low-energy CID and MALDI post-source decay for determination of O-glycosylation sites in MUC4 peptides
✍ Scribed by Alving, Kim; Körner, Roman; Paulsen, Hans; Peter-Katalinic, Jasna
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 175 KB
- Volume
- 33
- Category
- Article
- ISSN
- 1076-5174
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✦ Synopsis
Mucins are glycoproteins containing from one to several hundreds of carbohydrate chains attached to the peptide by O-glycosidic linkages between N-acetylgalactosamine (GalNAc) and either serine (Ser) or threonine (Thr). Apomucin proteins are encoded by di †erent mucin genes (MUC1-8) and show in general tandem repeats in which the carbohydrate chains can be highly clustered. The glycosylation density of human mucins seems to be related to some type of cancer. Fast atom-bombardment mass spectrometry (FAB-MS) has previously been used for the determination of O-glycosylation sites in combination with b-elimination. In this contribution, a comparative study by electrospray (ESI)-MS/MS and post-source decay (PSD)-MALDI-MS is presented. Nanospray-ESI-MS/MS experiments were performed using two analyzers, a triple quadrupole and a quadrupole ion trap. A series of O-GalNAc substituted synthetic MUC4 fragments with blocked N-and C-termini, and their nonglycosylated counterparts were investigated. In the sequence analysis of di †erently glycosylated and nonglycosylated MUC4 decapeptides by MALDI-PSD-MS, the spectra were dominated by b-and y-type ions. Substantial and structureunrelated loss of the sugar from molecular [ M + H ] ' and fragment ions was generally observed. The single GalNAc attachment site out of three to Ðve potential substitution Ser/Thr sites was identiÐed by attribution of Nand C-terminal fragment ions still carrying the GalNAc moiety. Similar fragmentation patterns were produced by nanospray ESI-low-energy CID on a Q3 instrument using Ar as a collision gas or on ion trap CID using He as a collision gas. Less abundant sequence ions were obtained from CID experiments on sodiated molecular [ M + Na ] ' ions, which required much higher energy.