Sialic acid linkage differentiation of glycopeptides using capillary electrophoresis - electrospray ionization - mass spectrometry

Abstract

Sialylation is a glycosylation feature that occurs in different linkages at the non-reducing end of a glycan moiety, the linkage isomers are often differentially associated with various biological processes. Due to very similar physico-chemical properties, the separation of isomeric sialylated glycopeptides remains challenging but of utmost importance in the biomedicine and biotechnology, including biomarker discovery, glyco-engineering and biopharmaceutical characterization. This study presents the implementation of a high-resolution separation platform based on capillary electrophoresis - mass spectrometry (CE-MS) allowing for the selective analysis of α2,3- and α2,6-sialylated glycopeptides. These differentially linked glycopeptides showed an identical fragmentation pattern (collision induced dissociation) but different electrophoretic mobilities, allowing for baseline separation of the different linkages without the need for an extensive sample preparation. The different migration behavior between the two moieties was found to correlate with differences in pK_a values. Using a novel methodology adapted from the so-called internal standard CE approach, a relative difference of 3.4·10^-2 in pK_a unit was determined. This approach was applied for the analysis of tryptic glycopeptides of prostate specific antigen, which shows highly complex and heterogeneous glycosylation. The developed platform therefore appears attractive for the identification of differentially linked sialic acids that may be related to pathological conditions.

Figure 1

Extracted ion electropherograms (EIEs) of IgGmAb1 and IVIgG1 glycopeptides obtained with CE–ESI-MS after targeted alignment. (A) EIEs of IgGmAb1 glycopeptides derived from CHO cells, (B) EIEs of IVIgG1 retrieved from human plasma and (C) EIEs of a co-injection of IgGmAb1 and IVIgG1. The “PEP” label illustrates the tryptic peptide sequence EEQYNSTYR to which the glycan is attached.

Figure 2

MALDI-TOF-MS spectrum of PNGase F released PSA N-glycans treated with ethyl esterification. A total of 37 different glycans were identified (not all data shown, a complete overview can be found in Table S-3, Supplementary Information).

Figure 3

CE–ESI-MS analysis of PSA tryptic (glyco)peptides. (A) Representative base peak electropherogram observed for a tryptic digest of PSA. Based on the electrophoretic separation, three distinct clusters were defined. (B) Cluster with only non-sialylated glycopeptides of PSA. (C) Cluster containing mono-sialylated glycopeptides of PSA. (D) Cluster with di-sialylated glycopeptides of PSA. A total of 75 different glycopeptides were identified (not all data shown, a complete overview can be found in Table S-3, Supplementary Information). Peaks with a star (*) were not assigned. The “PEP” label illustrates the tryptic peptide sequence N ₆₉K to which the glycan is attached.

Figure 4

CE–ESI-MS analysis of PSA with and without α2,3-sialidase treatment using CE–ESI-MS after targeted alignment. (A) EIEs of the non-sialylated glycopeptide (H5N4F1), the mono-sialylated glycopeptides (H5N4F1S1) and the di-sialylated glycopeptides (H5N4F1S2) of PSA prior to exoglycosidase treatment and (B) after exoglycosidase treatment.