Sialic acid linkage-specific permethylation for improved profiling of protein glycosylation by MALDI-TOF MS

Abstract

Protein glycosylation mediates a wide range of cellular processes, affecting development and disease in mammals. Deciphering the "glycocodes" requires rapid, sensitive and in-depth characterization of diverse glycan structures derived from biological samples. In this study, we described a two-step derivatization strategy termed linkage-specific sialic acid permethylation (SSAP) consisting of dimethylamination and permethylation for the improved profiling of glycosylation by matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (MS). High linkage-specificity (∼99%) of SSAP to both the two most common forms of sialic acid, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), permitted direct discrimination of α2,3- and α2,6-linked sialic acids in MALDI-TOF MS. The enhanced intensity (>10-fold) and increased detection limit (>10-fold) of derivatized glycans were valued for sensitive glycomics. Moreover, the good compatibility and reaction efficiency of the two steps of SSAP allowed rapid sample preparation (<2 h), benefiting robust analysis of glycans in a high-throughput manner. The SSAP strategy was further applied to investigate the protein glycosylation of human serum associated with rheumatoid arthritis (RA). It was demonstrated that the relative abundances of individual glycans were different in RA negative and RA positive samples, and meanwhile the RA patient/control ratios of both α2,3- and α2,6-sialylated glycans tended to elevate accompanied with the increase of sialylation. Those findings of the glycosylation changes occurred in human serum protein may contribute to the diagnosis of RA. Herein, SSAP derivatization combined with MALDI-TOF MS exhibits unique advantages for glycomic analysis and shows potential in glycosylation profiling of therapeutic proteins and clinical glycan biomarker discovery.

Keywords: Glycans; Glycosylation; MALDI-TOF MS; Rheumatoid arthritis; Sialic acid linkage-specific permethylation.

Fig. 1

Reaction scheme for the linkage-specific permethylation of N-acetylneuraminic and N-glycolylneuraminic acid (Neu5Ac and Neu5Gc) glycans. (A) α2,3-linked sialic acid reacts with the penultimate galactose forming a lactone which is subsequently recovered and permethylated. (B) α2,6-linked sialic acid is dimethylamidated and permethylated.

Fig. 2

SSAP derivatization and MALDI-TOF-MS analysis of sialylated N-glycan isomers in reflectron positive mode. (A, B) and (C, D) isomeric glycan terminated with a single Neu5Ac residue. (E, F) isomeric glycan terminated with two neighbouring Neu5Ac residues. (G, H) isomeric glycan attached with fucose and terminated with two Neu5Ac residues. All the MS peaks were detected as [M + Na]⁺ species. The nomenclature of used glycans refers to literature [30].

Fig. 3

Specificity analysis of SSAP derivatization based on the MALDI-TOF-MS intensity of four pairs of sialylated glycan isomers. The derivatization specificity of each glycan was calculated from three replications and showed as mean ± standard deviation.

Fig. 4

MALDI-TOF-MS analysis of (A) sialylated glycan derivatized via dimethylamidation and (B) sialylated glycan derivatized via SSAP in reflectron positive mode. All the MS peaks were detected as [M + Na]⁺ species.

Fig. 5

MALDI-TOF-MS/MS analysis of sialylated glycan isomers derivatized by SSAP. (A) MALDI-TOF-MS/MS analysis of derivatized N122 ([M + Na]⁺ precursor, m/z 2431.25). (B) MALDI-TOF-MS/MS analysis of derivatized N123 ([M + Na]⁺ precursor, m/z 2444.28).

Fig. 6

MALDI-TOF-MS analysis of SSAP derivatized N-glycans from (A) 5 μg of human IgG and (B) 5 μg of rat IgG in reflectron positive mode. All the MS peaks were detected as [M + Na]⁺ species.

Fig. 7

Bar charts representing the relative intensities of the MALDI-TOF-MS signals for the different N-glycan species detected in the serum of RA controls and RA patients. The experiment was performed three times on separate days. The relative intensities of glycan species were normalized to the overall sum of intensities of corresponding samples and shown as mean + standard deviation. Abbreviations used are hexose (H), N-acetylhexosamine (N), fucose (F) and N-acetylneuraminic acid with α2,3-linkage (S), and N-acetylneuraminic acid with α2,6-linkage (D).

Fig. 8

Bar chart demonstrating the changes in patient/control ratios of different types of sialylated N-glycans derived from RA serum. (A) Patient/control ratios of grouped SnDm types of glycans. (B) Patient/control ratios of grouped Sx and Dy types of glycans. S and D represent α2,3-linked sialic acid and α2,6-linked sialic acid, respectively, and n, m, x, and y indicate the numbers of sialic acid residues. The results were obtained from three independent experiments.