Assignment and quantification of 2-aminopyridine derivatized oligosaccharide isomers coeluted on reversed-phase HPLC/MS by MSn spectral library.
2-Aminopyridine (PA)-derivatized oligosaccharides from IgG were analyzed by using reversed-phase HPLC/mass spectrometry (RP-HPLC/MS) and a MS(n) spectral library, in particular, focusing on two pairs of isomers incompletely separated or coeluted in chromatograms. We previously reported that MS(n) spectral matching considering both major fragment ions (m/z) and intensities is useful and applicable to the structural assignment of PA-oligosaccharide isomers. In this study, MS(n) spectral matching based on the MS(n) spectral library was applied to the assignment of these PA-oligosaccharide isomers in IgG. Its usefulness was investigated by comparing it to the conventional two-dimensional mapping method based on retention time indexes. Specifically, we focus on the assignment and quantification of the isomers, which are coeluted in chromatograms. From this, we propose a new method using MS(n) spectral matching and the working curve on which are plotted the relative intensities of selected fragment ions in their MS(2) spectra versus various mixtures of the isomers. This new method demonstrated that the obtained quantities coincide very well with those estimated after separating by a combination of lectin and reversed-phase columns. This means that separation by RP-HPLC/MS is greatly simplified because complete separation of the isomers is no longer required. Application of this new method was tested by using the two other pairs of fucosylated and nonfucosylated PA-oligosaccharides from IgG. The results showed that this method works for them as well.
Takegawa
Yasuhiro
Y
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 001-0021, Japan.
Deguchi
Kisaburo
K
Ito
Shinya
S
Yoshioka
Shinji
S
Sano
Akihiro
A
Yoshinari
Kiyomi
K
Kobayashi
Kinya
K
Nakagawa
Hiroaki
H
Monde
Kenji
K
Nishimura
Shin-Ichiro
S
D000631:Aminopyridines / Q000032:analysis* / Q000737:chemistry; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005643:Fucose / Q000031:analogs & derivatives; D007074:Immunoglobulin G / Q000737:chemistry; D007536:Isomerism; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results
D000631:Aminopyridines; D007074:Immunoglobulin G; D009844:Oligosaccharides; D005643:Fucose