Identification of structurally closely related monosaccharide and disaccharide isomers by PMP labeling in conjunction with IM-MS/MS

Abstract

It remains particularly difficult for gaining unambiguous information on anomer, linkage, and position isomers of oligosaccharides using conventional mass spectrometry (MS) methods. In our laboratory, an ion mobility (IM) shift strategy was employed to improve confidence in the identification of structurally closely related disaccharide and monosaccharide isomers using IMMS. Higher separation between structural isomers was achieved using 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization in comparison with phenylhydrazine (PHN) derivatization. Furthermore, the combination of pre-IM fragmentation of PMP derivatives provided sufficient resolution to separate the isomers not resolved in the IMMS. To chart the structural variation observed in IMMS, the collision cross sections (CCSs) for the corresponding ions were measured. We analyzed nine disaccharide and three monosaccharide isomers that differ in composition, linkages, or configuration. Our data show that coexisting carbohydrate isomers can be identified by the PMP labeling technique in conjunction with ion-mobility separation and tandem mass spectrometry. The practical application of this rapid and effective method that requires only small amounts of sample is demonstrated by the successful analysis of water-soluble ginseng extract. This demonstrated the potential of this method to measure a variety of heterogeneous sample mixtures, which may have an important impact on the field of glycomics.

Figure 1. Structures for the 10 disaccharides and 4 monosaccharides used in this study.

Figure 2

A plot of CCS vs. m/z for (a) [M + Na]⁺ of underivatized carbohydrates, and (b) [M + Na]⁺ of PHN- and (c) [M + H]⁺ of PMP-derivatized carbohydrate isomers. RE (relative error) <5%. Refer to Table 1 for tabulated values.

Figure 3. Overall mobility spectra of the 9 PMP-derivatized disaccharide isomers and 3 PMP-derivatized monosaccharide isomers.

All mobility spectra of the disaccharides and monosaccharides were extracted for protonated ions at m/z 673.27 and 511.22, respectively. The arrival times are from three individual measurements, and deviation is ± 0.01 ms.

Figure 4. IM-MS/MS of the protonated PMP derivative ions of the three groups of disaccharide isomers.

(a) The mobility spectra were extracted for protonated product ions at m/z 499.19. (b) The mobility spectra were extracted for sodiated product ions at m/z 521.17. The arrival times are from three individual measurements, and deviation is ± 0.01 ms.

Figure 5

(a) Overall mobility spectra of the mixture of 9 PMP-derivatized disaccharide isomers and 3 PMP-derivatized monosaccharide isomers. All mobility spectra of the disaccharides and monosaccharides were extracted for protonated ions at m/z 673.27 and 511.22, respectively. (b) IM-MS/MS of the six unresolved PMP-disaccharide derivatives (kojibiose, nigerose, lactose, sophoros, cellobiose, and gentiobiose) unlabelled in the upper spectra. The mobility spectra were extracted for the product ions at m/z 499.19.

Figure 6

ATDs of (a) WGOS-1 and WGOS-2, (b) sucrose, and (c) glucose in the left column. ATDs of protonated PMP derivatives of (d) WGOS-1 and WGS-2, (e) glucose, (f) fructose, and (g) maltose in the right column. The red traces in Figure d and g are the drift time plots of fragment ions from the ions at m/z 673.27. The arrival times are from three individual measurements, and deviation is ± 0.01 ms.