title: Development of novel active acceptors possessing a positively charged structure for the transglycosylation reaction with Endo-M and their application to oligosaccharide analysis.
abstract: With Boc-Asn-GlcNAc as a basic structure, four permanently positively charged kinds of new acceptors (GP-Boc-Asn-GlcNAc, GT-Boc-Asn-GlcNAc, HMP-Boc-Asn-GlcNAc, MPDPZ-Boc-Asn-GlcNAc) and five kinds of similar structure acceptors (2-PA-Boc-Asn-GlcNAc, 3-PA-Boc-Asn-GlcNAc, 4-PA-Boc-Asn-GlcNAc, HP-Boc-Asn-GlcNAc, PDPZ-Boc-Asn-GlcNAc) were synthesized as acceptors for the resolution of oligosaccharides in glycopeptides. The synthesized acceptors enzymatically reacted with Disialo-Asn (donor) in the presence of Endo-M. The reaction yields of each transglycosylation product were not obvious, because we do not have all the authentic Disialo-Asn-Boc-acceptors. Therefore, we used the peak area of the transglycosylation product detected by mass spectrometry and evaluated the utility of each acceptor. Among the Boc-Asn-GlcNAc acceptors, the positively charged MPDPZ derivative peak area was the highest, MPDPZ-Boc-Asn-GlcNAc with a positively charged structure showed about a 2.2 times greater sensitivity of the transglycosylation product compared to the conventional fluorescence acceptor DBD-PZ-Boc-Asn-GlcNAc. As a result, the MPDPZ-Boc-Asn-GlcNAc acceptor was suitable for the transglycosylation reaction with Endo-M. The development of a qualitative determination method for the N-linked oligosaccharides in glycoproteins was attempted by combination of the transglycosylation reaction and semi-micro high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC/ESI-QTOF-MS/MS). The asparaginyl-oligosaccharides in glycoproteins, liberated by treatment with Pronase E, were separated, purified and labeled with positively charged MPDPZ. The resulting derivatives were separated by a semi-micro HPLC system. The eluted N-linked oligosaccharide derivatives were then introduced into a QTOF-MS instrument and sensitively detected in the ESI(+) mode. Various fragment ions based on the carbohydrate units appeared in the MS/MS spectra. Among the peaks, m/z 782.37 corresponding to MPDPZ-Boc-Asn-GlcNAc is the most important one for identifying the asparaginyl-oligosaccharides. Disialo-Asn-Boc-MPDPZ was easily identified by the selected-ion chromatogram at m/z 782.37 by MS/MS detection. Therefore, the identification of N-linked oligosaccharides in glycoproteins seems to be possible by the proposed semi-micro HPLC separations followed by the QTOF-MS/MS detection. Furthermore, several oligosaccharides in ovalbumin and ribonuclease B were successfully identified by the proposed procedure.
authors.affiliation: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, and Global COE Program, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. junzhe@u-shizuoka-ken.ac.jp
authors.forename: Jun Zhe
authors.initials: JZ
authors.lastname: Min
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Yuki
authors.initials: Y
authors.lastname: Tomiyasu
authors.forename: Dongri
authors.initials: D
authors.lastname: Jin
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Higashi
authors.forename: Yong-Ill
authors.initials: YI
authors.lastname: Lee
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Toyo'oka
mesh_terms: D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism*; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D010047:Ovalbumin / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D009844:Oligosaccharides; D010047:Ovalbumin; D012260:Ribonucleases; C029600:ribonuclease B; D006596:Hexosaminidases