Profiling pneumococcal type 3-derived oligosaccharides by high resolution liquid chromatography-tandem mass spectrometry.
Pneumococcal type-3 polysaccharide (Pn3P) is considered a major target for the development of a human vaccine to protect against Streptococcus pneumoniae infection. Thus, it is critical to develop methods for the preparation and analysis of Pn3P-derived oligosaccharides to better understand its immunological properties. In this paper, we profile oligosaccharides, generated by the free radical depolymerization of Pn3P, using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Hydrophilic liquid interaction chromatography (HILIC)-mass spectrometry (MS) revealed a series of oligosaccharides with an even- and odd-number of saccharide residues, ranging from monosaccharide, degree of polymerization (dp1) to large oligosaccharides up to dp 20, generated by free radical depolymerization. Isomers of oligosaccharides with an even number of sugar residues were easily separated on a HILIC column, and their sequences could be distinguished by comparing MS/MS of these oligosaccharides and their reduced alditols. Fluorescent labeling with 2-aminoacridone (AMAC) followed by reversed phase (RP)-LC-MS/MS was applied to analyze and sequence poorly separated product mixtures, as RP-LC affords higher resolution of AMAC-labeled oligosaccharides than does HILIC-based separation. The present methodology can be potentially applied to profiling other capsular polysaccharides.
College of Food Science and Technology, Ocean University of China, Qingdao, Shandong 266003, China; Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Guoyun
G
Li
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Lingyun
L
Li
College of Food Science and Technology, Ocean University of China, Qingdao, Shandong 266003, China.
Changhu
C
Xue
Department of Biochemistry and Molecular Biology, Center for Molecular Medicine, and Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA.
Dustin
D
Middleton
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. Electronic address: linhar@rpi.edu.
Robert J
RJ
Linhardt
Department of Biochemistry and Molecular Biology, Center for Molecular Medicine, and Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA. Electronic address: avci@uga.edu.
Fikri Y
FY
Avci
D000609:Aminoacridines / Q000737:chemistry; D002623:Chemistry Techniques, Analytical; D002853:Chromatography, Liquid; D057927:Hydrophobic and Hydrophilic Interactions; D007536:Isomerism; D009844:Oligosaccharides / Q000737:chemistry*; D013296:Streptococcus pneumoniae / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
D000609:Aminoacridines; D009844:Oligosaccharides; C072515:2-aminoacridone
Fluorescent labeling; Free radical depolymerization; Isomeric oligosaccharides; Liquid chromatography–mass spectrometry; Pneumococcal type 3 polysaccharide