Hiroyuki Satake

An Efficient Approach for the Characterization of Mucin‐Type Glycopeptides: The Effect of O‐Glycosylation on the Conformation of Synthetic Mucin Peptides

Despite the growing importance of mucin core O-glycosylation in many biological processes including the protection of epithelial cell surfaces, the immune response, cell adhesion, inflammation, and tumorigenesis/metastasis, the regulation mechanism and conformational significance of the multiple introduction of α-GalNAc residues by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) remains unclear. Here we report an efficient approach by combining MS and NMR spectroscopy that allows for the identification of O-glycosylation site(s) and the effect of O-glycosylation on the peptide backbone structures during enzymatic mucin domain assembly by using an isoform UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T2 (ppGalNAcT2) in vitro. An electron-capture dissociation device in a linear radio-frequency quadrupole ion trap (RFQ-ECD) combined with a time-of-flight (TOF) mass spectrometer was employed for the identification of Thr/Ser residues occupied by α-GalNAc branching among multiple and potential O-glycosylation sites in the tandem repeats of human mucin glycoproteins MUC4 (Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp) and MUC5AC (Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly). In the present study, O-glycosylation was initiated specifically at Thr10 in naked MUC4 peptide and additional introduction of α-GalNAc proceeded preferentially but randomly at three other Thr residues to afford densely glycosylated MUC4 containing six α-GalNAc residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15. On the contrary, O-glycosylation of naked MUC5AC peptide occurred predominantly at consecutive Thr residues and led to MUC5AC with four α-GalNAc residues at Thr2, Thr3, Thr7, and Thr8. The solution structures determined by NMR spectroscopic studies elicited that the preferential introduction of α-GalNAc at Thr10 of MUC4 stabilizes specifically a β-like extended backbone structure at this area, whereas other synthetic models with a single α-GalNAc residue at Thr1, Thr6, or Thr15 did not exhibit any converged three-dimensional structure at the proximal peptide moiety. Such conformational impact on the underlying peptides was proved to be remarkable in the glycosylation at the consecutive Thr residues of MUC5AC.

Glycopeptide identification using liquid-chromatography-compatible hot electron capture dissociation in a radio-frequency-quadrupole ion trap.

We developed a liquid chromatography (LC) compatible electron capture dissociation (ECD) mass spectrometer for glycoproteomics, with which ECD and hot ECD (HECD) experiments can be flexibly switched by quickly changing the electron energy without further tuning of the mass spectrometer. Desialylated glycopeptides were dissociated well in both ECD and HECD experiments. For sialylated glycopeptides, on the other hand, ECD with electron energy higher than 4 eV showed significantly higher sequence coverage than that with an electron energy of 0.2 eV. A nano LC system was coupled to our ECD mass spectrometer to investigate N-linked glycopeptides from lysylendopeptidase (Lys-C) digests of human transferrin. ECD spectra at multiple electron energies of 0.2, 5.0, and 9.0 eV were obtained for each targeting precursor ion in a single LC injection. Glycopeptides with a sialylated bi-, tri-, or tetra-antennary complex N-glycan were identified with high sequence coverage by HECD. Glycopeptides with tri- or tetra-antennary N-glycans have seldom been analyzed by ECD or ETD before this report. We also found that a preferential dissociation of nonreducing termini of glycans in glycopeptides by ECD and HECD.

Elucidating the sequence of intact bioactive peptides by using electron capture dissociation and hot electron capture dissociation in a linear radio-frequency quadrupole ion trap.

RATIONALE Electron capture dissociation (ECD) is useful tool for sequencing of peptides and proteins with post-translational modifications. To increase the sequence coverage for peptides and proteins, it is important to develop ECD device with high fragmentation efficiency. METHODS Sequence analysis of intact undigested bioactive peptides (3000-5000 Da) was performed by use of electron capture dissociation (rf-ECD) and collision-induced dissociation (CID) in a linear radio-frequency quadrupole ion trap that was coupled to a time-of-flight mass spectrometer. We applied rf-ECD, hot rf-ECD (rf-ECD with high electron energy), and CID for intact bioactive peptide ions of various charge states and evaluated the sequence coverage of their fragment spectra. RESULTS Hot rf-ECD produced a higher number of c- and z-type fragment ions of modified peptide ions as electron energy increased in lower charged peptide ions, and sequence coverage greater than 80% was obtained compared with the CID case (40-80%). CONCLUSIONS The result indicates that intact bioactive modified peptides (Ghrelin, ANP) were correctly identified by use of hot rf-ECD.