A glycomics platform for the analysis of permethylated oligosaccharide alditols

Abstract

This communication reports the development of an LC/MS platform for the analysis of permethylated oligosaccharide alditols that, for the first time, demonstrates routine online oligosaccharide isomer separation of these compounds before introduction into the mass spectrometer. The method leverages a high-resolution liquid chromatography system with the superior fragmentation pattern characteristics of permethylated oligosaccharide alditols that are dissociated under low-energy collision conditions using quadrupole orthogonal time-of-flight (QoTOF) instrumentation and up to pseudo MS(3) mass spectrometry. Glycoforms, including isomers, are readily identified and their structures assigned. The isomer-specific spectra include highly informative cross-ring and elimination fragments, branch position specific signatures, and glycosidic bond fragments, thus facilitating linkage, branch, and sequence assignment. The method is sensitive and can be applied using as little as 40 fmol of derivatized oligosaccharide. Because permethylation renders oligosaccharides nearly chemically equivalent in the mass spectrometer, the method is semiquantitative and, in this regard, is comparable to methods reported using high field NMR and capillary electrophoresis. In this postgenomic age, the importance of glycosylation in biological processes has become clear. The nature of many of the important questions in glycomics is such that sample material is often extremely limited, thus necessitating the development of highly sensitive methods for rigorous structural assignment of the oligosaccharides in complex mixtures. The glycomics platform presented here fulfills these criteria and should lead to more facile glycomics analyses.

Figure 1

LC/MS analysis of permethylated maltooligosaccharide alditols. A, Extracted ion chromatograms of permethylated oligosaccharide alditol maltooligosaccharide series Glc_4-8. The Glc₄, Glc₅ Glc₆, Glc₇ [M+Na]⁺ ions, Glc₈ [M+2Na]²⁺ were detected at m/z 901.45, 1105.56, 1309.66, 1513.76 and 870.43 respectively. Integrated peak areas are proportional to abundances of the glycoforms. B and C, fragment origin and CID QoTOF MS/MS spectra of Glc5, [M+Na]⁺ m/z 1105.56. Fragmentation nomenclature is that of Domon and Costello . All fragments contain sodium. Monosaccharide component symbols are those suggested by Varki et al . Key ions are indicated. The inset in C demonstrates that distinct B, Z, C and Z ions are produced from the reduced and permethylated Hexose oligosaccharide.

Figure 2

Extracted ion chromatogram of permethylated oligosaccharide alditols Man_5-9GlcNAc₂: A, a stacked plot of molecular ions for Man_5-9GlcNAc₂is shown; B, Three partially overlapping peaks are observed for Man₇GlcNAc₂ detected at [M+2Na]²⁺ m/z 1013.49 (bottom) and are labeled as D1, D2, and D3 and three are observed for Man₈GlcNAc₂ detected at [M+2Na]²⁺ m/z 1115.55 and are labeled as D1D2, D2D3 and D1D3 (top); C, Selected ion chromatogram of Man₉GlcNAc₂ produced from approximately 160 fmol. The molecular ion region of the mass spectrum is shown in the inset. See text for details concerning nomenclature.

Figure 3

CID QoTOF MS/MS spectra of Man₇GlcNAc₂ D1, D2, and D3 isomers. Major product ions, derived from [M+2Na]²⁺ m/z 1013.49 are indicated in each spectrum and their origins depicted to the structural representations to the right of each spectrum. Spectra and structural diagrams are shown in the order of their elution: A, Man₇GlcNAc₂ D2; B, Man₇GlcNAc₂ D3, and C, Man₇GlcNAc₂ D1. All fragments contain sodium.

Figure 4

LC/MS analysis of articular cartilage decorin N-glycans. A, The extracted ion chromatogram of articular cartilage decorin dHex₁Hex₄HexNAc₄ permethylated oligosaccharide alditol is shown. Two well resolved peaks are observed for [M+2Na]²⁺ m/z 1039.56 and are labeled as A and B. B and C, CID QoTOF MS/MS spectra of articular cartilage decorin dHex₁Hex₄HexNAc₄ permethylated oligosaccharide alditol isomers. The [M+2Na]²⁺ ions were isolated at m/z 1039.56. Key ions are indicated in each spectrum (A and C) and their origins are depicted in the structural representations to the right of each spectrum (B and C). All fragments contain sodium.

Figure 5

Trace ion chromatogram of reduced and permethylated C. elegans Hex₃HexNAc₁. Three major peaks are observed for [M+Na]⁺ m/z 942.55 and are labeled as A, B and C.

Figure 6

CID QoTOF MS/MS spectra of C. elegans Hex₃GalNAc₁ permethylated oligosaccharide alditol isomers isolated at [M+Na]⁺ m/z 942.55. The spectra of Peaks A, B and C are shown. Key ions are indicated in each spectrum and their origins and resultant linkage assignments are depicted in the structural representations to the right of each spectrum. Peaks B and C each contained two isomers, the lower arm of which was either Hex1,4Hex1,3- or Hex1,6Hex1,3-linked to the core GalNAc-ol. Specific structural assignments are based on pseudo MS³ spectrum shown in Figure 9. All fragments contain sodium. Monosaccharide identities are based on those of Varkii et al., [49] as in other figures except that hexoses that can be either Gal or Glc are represented as circles with an X.

Figure 7

Pseudo MS³ spectra of key product ions of Hex₃GalNAc₁ permethylated alditol isomers. A:³X₂ m/z 415.19 ion of Peak A, B; Z_1α m/z 502.26 ion for Peak B, C; Z_1α m/z 502.26 ion for Peak C, and D; ^1,4A_2α m/z 463.22 ion of Peak C. Proposed structures are shown above their spectra. In B and C, the Z_1α m/z 502.26 ion can be either a 1,4 or 1,6 glycosidic bond. See text for details. All fragments contain sodium.

Scheme I

Decomposition of the 1-4-linked disaccharide isolated at m/z 502.26. The proposed pathway for the formation of key fragment ions is shown.