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), permitting 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.

Identifying Sialylation Linkages at the Glycopeptide Level by Glycosyltransferase Labeling Assisted Mass Spectrometry (GLAMS)

“…For the distribution analysis of α2,3 or α2,6-sialylation ( Figure 5A ), only 71 distinct site-specific sialoglycopeptides contained pure α2,3-sialylation linkage, 186 sialoglycopeptides contained hybrid sialylation, and all the rest were uniquely 2,6-sialylated, which accounts for more than 77% of the identified sialoglycopeptides. This is consistent with the previous result that α2,6-sialylation is dominant in sialylated N-glycans released from human serum samples 54 . Taking a closer look at the distribution of sialylation levels, monosialylation is the major component in strictly α2,3 or α2,6-sialoglycopeptides, and 83% of the hybrid sialoglycopeptides are disialylated.…”

Advancing a High Throughput Glycotope-centric Glycomics Workflow Based on NAnoLC-MS2-product Dependent-MS3 ANAlysis of Permethylated Glycans*

“…On the other hand, structural features that will not directly yield a diagnostic ion can potentially be defined by incorporating additional chemo-enzymatic manipulation steps. For example, recent work by Jiang et el demonstrated that ␣2-3 and 2-6-sialic acids can be differentially derivatized by dimethylamidation followed by permethylation (25), resulting in 13 mass units difference between a methyl esterified and dimethyamidated sialic acid depending on whether it is 2-3 or 2-6-linked to Gal, respectively. This allows production of distinctive NeuAc ϩ and NeuAc-containing oxonium ions for a variety of ␣2-3/2-6-linked sialylated glycotopes to be distinguished and relatively quantified using our workflow.…”

Mass spectrometry for protein sialoglycosylation

“…However, the generated lactones suffer from the instability in water solution, resulting in poor reproducibility. In recent years, two‐step derivatization protocols have been applied to derivatize α2,3‐linked sialic acids to amides or esters instead of lactones . In the first step, α2,6‐linked sialic acids are efficiently derivatized by ammonium chloride, dimethylamine, or ethanol; whereas α2,3‐linked sialic acids form lactones.…”

“…In recent years, two-step derivatization protocols have been applied to derivatize α2,3-linked sialic acids to amides or esters instead of lactones. [257][258][259][260][261] In the first step, α2,6-linked sialic acids are efficiently derivatized by ammonium chloride, dimethylamine, or ethanol; whereas α2,3-linked sialic acids form lactones. In the second step, the lactones are regenerated and subsequently converted to amides or esters.…”