title: Highly sensitive derivatization reagents possessing positively charged structures for the determination of oligosaccharides in glycoproteins by high-performance liquid chromatography electrospray ionization tandem mass spectrometry.
abstract: We have developed three kinds of novel derivatization reagents (4-CEBTPP, 4-CBBTPP, 5-COTPP) with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge for resolution of the oligosaccharides in glycoprotein using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The synthesized reagents reacted with the sialylglycosylamine of the sialylglycopeptide after treatment by PNGase F. The final derivatives were analyzed by ESI-MS and sensitively detected in the selected reaction monitoring (SRM) mode. Furthermore, the limits of detection (S/N=3) on the SRM chromatograms were at the fmol level (30fmol). Therefore, we used the limit of detection of the reagent products detected by the SRM and evaluated the utility of each reagent. Among the reagents, the positively charged 4-CEBTPP derivative's peak area was the highest; 4-CEBTPP with a positively charged structure showed about a 20 times greater sensitivity for the glycosylamine of the SGP product compared to the conventional fluorescence reagent, Fmoc-Cl. In addition, various fragment ions based on the carbohydrate units also appeared in the MS/MS spectra. Among the fragment ions, m/z 627.37 (CE=40eV) corresponding to 4-CEBTPP-GlcNAc and m/z 120.09 (CE=100eV) corresponding to 4-CEBTPP are the most important ones for identifying the oligosaccharide. 4-CEBTPP-SGA was easily identified by the selected-ion chromatogram in the product ion scan (m/z 120.09) and in the precursor ion scan (m/z 627.37) by MS/MS detection. The derivatized analytes have a high ionization efficiency and they are detected with a high sensitivity in the electrospray ionization. The novel derivatization reagent with a multi-function provided a higher sensitivity for the oligosaccharide analysis, as well as a better specificity and feasibility. Furthermore, several oligosaccharides in fetuin and ribonuclease B were successfully identified by the proposed procedure.
authors: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy Yanbian University and Department of Pharmacy, Affiliated Western Hospital, Yanbian University, Yanji, 133002, Jilin Province, China; Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan. Electronic address: junzhemin23@163.com.
authors: Jun Zhe
authors: JZ
authors: Min
authors: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
authors: Keisuke
authors: K
authors: Nagai
authors: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy Yanbian University and Department of Pharmacy, Affiliated Western Hospital, Yanbian University, Yanji, 133002, Jilin Province, China.
authors: Qing
authors: Q
authors: Shi
authors: Division of Diabetes, Endocrinology and Metabolism, Department of Medicine Baylor, College of Medicine Houston, TX, 77030, USA.
authors: Wenjun
authors: W
authors: Zhou
authors: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.
authors: Kenichiro
authors: K
authors: Todoroki
authors: Laboratory of Clinical and Analytical Chemistry, College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga, 525-8577, Japan.
authors: Koichi
authors: K
authors: Inoue
authors: Department of Chemistry, Changwon National University, Changwon, 641-773, South Korea.
authors: Yong-Ill
authors: YI
authors: Lee
authors: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan. Electronic address: toyooka@u-shizuoka-ken.ac.jp.
authors: Toshimasa
authors: T
authors: Toyo'oka
mesh_terms: D002851:Chromatography, High Pressure Liquid; D006023:Glycoproteins / Q000737:chemistry*; D007202:Indicators and Reagents / Q000737:chemistry; D057230:Limit of Detection; D009844:Oligosaccharides / Q000032:analysis*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D012260:Ribonucleases / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D006023:Glycoproteins; D007202:Indicators and Reagents; D009844:Oligosaccharides; D009943:Organophosphorus Compounds; C061896:triphenylphosphine; D012260:Ribonucleases; C029600:ribonuclease B
keywords: Glycoprotein; LC-ESI–MS; N-linked glycan; Oligosaccharide; Positively charged; Triphenylphosphine