Identification of N-linked oligosaccharide labeled with 1-pyrenesulfonyl chloride by quadrupole time-of-flight tandem mass spectrometry after separation by micro- and nanoflow liquid chromatography.
The development of a qualitative determination method for the N-linked oligosaccharides in glycoproteins was attempted by the combination of micro- or nanoflow LC with Q-TOF-MS/MS. The asparaginyl-oligosaccharides in glycoproteins, liberated from the treatment of Pronase E, were separated, purified and labeled with 1-pyrenesulfonyl chloride (PSC). The resulting derivatives were separated by the microflow LC system utilizing a 0.5 mm diameter microcolumn or nanoflow LC system utilizing a 75 microm diameter chip column. The eluted N-linked oligosaccharide derivatives were then introduced into the Q-TOF-MS instrument and sensitively detected in the ESI(+) mode. Several factors (i.e. fragmentor, skimmer, Vcap voltages and collision energy) affecting the sensitivity of Q-TOF-MS/MS detection were optimized in both the micro- and nanoflow LC systems. Various fragment ions based on the carbohydrate units appeared on the MS/MS spectra. Among the peaks, m/z 600.16 corresponding to PSC-labeled Asp-HexNAc is the most important one to identify the asparaginyl-oligosaccharide. The N-linked oligosaccharides in the ovalbumin were easily identified by the selected-ion chromatogram at m/z 600.16 by the MS/MS detection. Therefore, the identification of N-linked oligosaccharides in glycoproteins seems to be possible by the proposed micro- and nanoflow LC separations followed by the Q-TOF-MS/MS detection.
Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, and Global COE Program, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Japan.
Jun Zhe
JZ
Min
Takamasa
T
Kurihara
Asuka
A
Hirata
Toshimasa
T
Toyo'oka
Shinsuke
S
Inagaki
D000117:Acetylglucosamine / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005690:Galactose / Q000032:analysis; D008358:Mannose / Q000032:analysis; D036103:Nanotechnology / Q000379:methods; D009844:Oligosaccharides / Q000032:analysis*; D011721:Pyrenes / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D009844:Oligosaccharides; D011721:Pyrenes; C104379:pyrene sulfonyl chloride; D008358:Mannose; D000117:Acetylglucosamine; D005690:Galactose