Assignment and Quantification of 2-Aminopyridine Derivatized Oligosaccharide Isomers Coeluted on Reversed-Phase HPLC/MS by MSⁿ Spectral Library

Abstract

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.

Discrimination of 16 structural isomers of fucosyl galactoside based on energy-resolved mass spectrometry

“…It was observed that compounds closely related structurally consisting of anomers and linkage isomers could be distinguished by CID spectra when multiple ions were obtained. This indicates that the spectral differences often observed for isomeric natural oligosaccharides are not just a coincidence, and also supports the usefulness of methods based on spectral matching [11][12][13][14][15][16][17]. A problem, however, is that this is true only when a sufficient number of distinct signals were collected for the comparison.…”

“…In most cases, there are differences in signal intensities, whereas in others, different fragment ions can be found. Accumulation of the spectral data in a database facilitates identification of oligosaccharides based on spectral matching [11][12][13][14][15][16][17]. An important problem-the elucidation of totally unknown structures that are not present in any databasewill still remain after the introduction of this efficient methodology.…”

Detection of altered N-glycan profiles in whole serum from rheumatoid arthritis patients

“…Following improvements in sensitivity or resolution, the method utilized here is applicable to other disease or oligosaccharide alterations. SSI-IT/MS can also be used to analyze sialyl oligosaccharides and distinguish isomers using MS n [12,13]; thus a combination of SSI-IT/MS and RP-HPLC should be widely applicable to N-glycan analysis.…”

“…Sonic spray ionization ion trap mass spectrometry (SSI-IT/MS) ionizes mildly with nitrogen 4 gas flow [12] and is applicable to quantitative analysis of neutral and sialic oligosaccharides in the negative ion mode [13,14]. Thus we combined SSI-IT and MS in order to compare N-glycan profiles from whole sera of healthy women and female RA patients.…”

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“…Weiskopf et al also demonstrated that the core pentasaccharide structure generated from a sialylated and an asialyl-N-glycan exhibit a similar fragment pattern in their MS 4 and MS 3 spectra (referred to as``MS nˆn gerprinting'') (28). The MS n spectral library methods for pyridylamino (PA) N-glycans proposed by Takegawa et al (30,31) and by Kameyama et al (7,32,33) also fall into this category. All of these similar approaches can be considered as versions of``MS n spectral matching.''…”