Accurate Identification of Isomeric Glycans by Trapped Ion Mobility Spectrometry-Electronic Excitation Dissociation Tandem Mass Spectrometry

Abstract

Ion mobility-mass spectrometry (IM-MS) has become a powerful tool for glycan structural characterization due to its ability to separate isomers and provide collision cross section (CCS) values that facilitate structural assignment. However, IM-based isomer analysis may be complicated by the presence of multiple gas-phase conformations of a single structure that not only increases difficulty in isomer separation but can also introduce the possibility for misinterpretation of conformers as isomers. Here, the ion mobility behavior of several sets of isomeric glycans, analyzed as their permethylated derivatives, in both nonreduced and reduced forms, was investigated by gated-trapped ion mobility spectrometry (G-TIMS). Notably, reducing-end reduction, commonly performed to remove anomerism-induced chromatographic peak splitting, did not eliminate the conformational heterogeneity of permethylated glycans in the gas phase. At a mobility resolving power of ∼100, 14 out of 22 structures showed more than one conformation. These results highlight the need to use IMS devices with high mobility resolving power for better separation of isomers and to acquire additional structural information that can differentiate isomers from conformers. Online electronic excitation dissociation (EED) MS/MS analysis of isomeric glycan mixtures following G-TIMS separation showed that EED can generate isomer-specific fragments while producing nearly identical tandem mass spectra for conformers, thus allowing confident identification of isomers with minimal evidence of any ambiguity resulting from the presence of conformers. G-TIMS EED MS/MS analysis of N-linked glycans released from ovalbumin revealed that several mobility features previously thought to arise from isomeric structures were conformers of a single structure. Finally, analysis of ovalbumin N-glycans from different sources showed that the G-TIMS EED MS/MS approach can accurately determine the batch-to-batch variations in glycosylation profiles at the isomer level, with confident assignment of each isomeric structure.

Figure 1.

SNFG (symbol nomenclature for glycans) and representations of the structures of glycans discussed in the text.

Figure 2.

Extracted ion mobiligrams (EIMs) of permethylated (a-e) and reduced and permethylated (f-j) LNFP I, II, III, V, and VI, [M+Na]⁺, respectively.

Figure 3.

G-TIMS EED MS/MS analysis of a mixture of permethylated LNFP II and III. (a) EIMs of the [M+Na]⁺ precursor ions and the fragment ions at m/z 442.204 and m/z 472.215; (b, c) EED spectra acquired at two elution voltages marked in (a). Isomer-diagnostic fragments are labeled in color.

Figure 4.

G-TIMS EED MS/MS analysis of a mixture of reduced and permethylated LNFP II and III. (a) EIMs of the [M+Na]⁺ precursor ions at m/z 1117.584 and two fragment ions at m/z 442.204 and m/z 472.215; (b-e) EED spectra acquired at four elution voltages marked in (a). Isomer-diagnostic fragments are labeled in color.

Figure 5.

G-TIMS EED MS/MS analysis of permethylated H₅N₄ glycans. (a-c) EIMs of the H₅N₄ glycans ([M+2Na]²⁺, m/z 1046.5122) released from chicken ovalbumin Batch 1, the bisected GlcNAc glycan standard (H₅N₄ isomer #1 in Figure 1), and H₅N₄ released from chicken ovalbumin Batch 2, respectively. (d) CCS values of H₅N₄ isomers measured by G-TIMS.

Figure 6.

(a-c) EED spectra of H₅N₄ ([M+2Na]²⁺, m/z 1046.512) from chicken ovalbumin Batch 1, acquired at the positions marked as 1, 2, and 3 in Figure 5a, respectively. (d) The EED spectrum of H₅N₄ from chicken ovalbumin Batch 2, acquired at position 4 in Figure 5c. Fragments discussed in the text are labeled in color.