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Development of novel active acceptors possessing a positively charged structure for the transglycosylation reaction with Endo‐M and their application to oligosaccharide analysis
Author(s) -
Min Jun Zhe,
Suzuki Yasumasa,
Tomiyasu Yuki,
Jin Dongri,
Higashi Tatsuya,
Lee YongIll,
Toyo'oka Toshimasa
Publication year - 2011
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.5190
Subject(s) - chemistry , oligosaccharide , combinatorial chemistry , stereochemistry , computational chemistry , organic chemistry
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. Copyright © 2011 John Wiley & Sons, Ltd.