High-Sensitivity Glycan Profiling of Blood-Derived Immunoglobulin G, Plasma, and Extracellular Vesicle Isolates with Capillary Zone Electrophoresis-Mass Spectrometry

Abstract

We developed a highly sensitive method for profiling of N-glycans released from proteins based on capillary zone electrophoresis coupled to electrospray ionization mass spectrometry (CZE-ESI-MS) and applied the technique to glycan analysis of plasma and blood-derived isolates. The combination of dopant-enriched nitrogen (DEN)-gas introduced into the nanoelectrospray microenvironment with optimized ionization, desolvation, and CZE-MS conditions improved the detection sensitivity up to ∼100-fold, as directly compared to the conventional mode of instrument operation through peak intensity measurements. Analyses without supplemental pressure increased the resolution ∼7-fold in the separation of closely related and isobaric glycans. The developed method was evaluated for qualitative and quantitative glycan profiling of three types of blood isolates: plasma, total serum immunoglobulin G (IgG), and total plasma extracellular vesicles (EVs). The comparative glycan analysis of IgG and EV isolates and total plasma was conducted for the first time and resulted in detection of >200, >400, and >500 N-glycans for injected sample amounts equivalent to <500 nL of blood. Structural CZE-MS² analysis resulted in the identification of highly diverse glycans, assignment of α-2,6-linked sialic acids, and differentiation of positional isomers. Unmatched depth of N-glycan profiling was achieved compared to previously reported methods for the analysis of minute amounts of similar complexity blood isolates.

Figure 1.

Comparison of MS signal intensity levels for glycan peaks in CZE-MS analyses (n = 3) of APTS-labeled dextran ladder standards at the evaluated conditions. (A, B) Comparison of different nESI microenvironment control modes: (1) a conventional mode of instrument operation; (2) a purified air (ABIRD); and (3) a nitrogen gas enriched with IPA. (C, D) Optimization of the MS ion transfer tube (ITT) temperature, without applying any additional control of the nESI microenvironment.

Figure 2.

Comparison of extracted ion electropherograms (EIEs, panels (A)–(K)) and MS spectra corresponding to DP7 (C–L) in CZE-MS analyses (n = 3) of APTS-labeled dextran ladder standards DP4–DP29 at the following evaluated conditions: (A–C) ITT at 110 °C; (D–F) ITT at 150 °C; (G–I) ITT at 150 °C and ISCID at 70 eV; and (J–L) ITT at 150 °C, ISCID at 70 eV, and DEN-gas with IPA.

Figure 3.

Effect of experimental conditions on CZE-MS-based glycan profiling. (A, B) CZE-MS analyses (n = 3) of human serum IgG at five different conditions: (1) conventional mode of instrument operation (ITT at 110 °C); (2) purified air (ABIRD) (ITT at 110 °C); (3) nitrogen gas enriched with IPA (ITT at 110 °C); (4) nitrogen gas enriched with IPA (ITT at 150 °C); and (5) nitrogen gas enriched with IPA (ITT at 150 °C) and ISCID at 70 eV. (C, D) Ion density maps (intensity level: 3 × 10⁵) of the CZE-MS analyses of human serum IgG before (C) and after (D) method optimization.

Figure 4.

Effect of experimental conditions on the resolution in CZE-MS separation of selected APTS-labeled glycans. EIEs of G2FS1, G0F, G1F, and G2F IgG glycans analyzed at the following conditions: (A) conventional mode of instrument operation (ITT at 110 °C) and supplemental pressure of 5 psi, (B, C) nitrogen gas enriched with IPA (ITT at 150 °C) and ISCID at 70 eV with (B) or without (C) supplemental pressure of 5 psi. For (A) and (B), the supplemental pressure was applied 18 min after the beginning of the CZE-MS run.

Figure 5.

Differential N-glycan profiling of blood-derived samples. (A, B) Results of Euclidean distance-based hierarchical clustering of N-glycan quantitative profiles detected in human serum IgG, human plasma total EV isolate, and total human plasma. (A) Circular heatmap showing clustered groups of all detected N-glycans. (B) Heatmap cluster showing N-glycans detected in all three sample types. Red, yellow, and light blue colors correspond to high, medium, and low relative abundances based on the N-glycan signal intensities. N-glycans that are not detected in the samples are highlighted in dark blue. (C–H) Distribution of different types of N-glycans based on the number of identifications in the above-mentioned biological samples. (C–E) Fucosylated N-glycans. (F–H) Neu5Ac-containing N-glycans.

Figure 6.

Examples of characteristic tandem mass spectra of APTS-labeled FA2G2S2 (A) and FA2BG2S2 (B) detected in human serum IgG and human plasma EV isolates. The MS²-based structural characterization was performed by selecting the [M − 2H]²⁻ molecular ion at m/z 1403.89 and the [M − 3H]³⁻ molecular ion at m/z 1003.29, respectively, as precursor ions. Fragment ions are annotated based on the Domon and Costello nomenclature. Blue square, GlcNAc; red triangle, Fuc; green circle, Man; yellow circle, Gal; purple diamond; Neu5Ac; and orange star, APTS. Symbol Z relates to cross-ring fragmentation. Only the most intense/relevant fragments are annotated in the shown spectra.