Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti4+-SPE enrichment for mass spectrometric analysis

Abstract

N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti(4+)-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti(4+)-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CV<15%) as well as high sensitivity (LOD at fmol level). At last, the proposed method was successfully applied in the profiling of N-glycome in human serum, in which a total of 31 N-glycan masses were identified.

Integrated Pipeline of Isotopic Labeling and Selective Enriching for Quantitative Analysis of N-Glycome by Mass Spectrometry

“…The enrichment of N -glycans before MS analysis becomes a prerequisite to avoid suppression caused by peptides and other substances that are easily ionized . Lectin, porous graphitized carbon, , hydrophilic interaction chromatography, size exclusion chromatography, hydrazide chemistry, and tagging-assisted enrichment methods , were utilized to purify the N -glycans. Among them, the tagging-assisted methods that introduce an affinity tag at the reducing end of the glycan and take advantage of the interaction between the tag and the solid-phase material to selectively capture the N -glycans show their superior selectivity.…”

Discovery of Non-invasive Glycan Biomarkers for Detection and Surveillance of Gastric Cancer

“…MS is emerging as an enabling technology in the field of glycomics. Meanwhile, ongoing efforts into sample preparation strategies compatible with MS were taken to improve analytical results, such as different enrichment and derivatization methods 17 , 26 , 27 . In this work, ethyl esterification derivatization of glycans was employed to increase their stability for MS analysis.…”

Mass Spectrometry Approaches to Glycomic and Glycoproteomic Analyses

“…93 The most hydrophobic reagent, n-Pr 2 N, was found to yield the most enhanced signal and applied to N-glycans released from human serum. Other new labels that can facilitate the glycan enrichment after derivatization were also investigated such as 4-aminophenylphosphate tag combined with Ti 4+ -SPE 94 and heptadecafluoroundecylamine tag combined with fluorous SPE. 95 Jiang et al developed a global solid-phase approach for the reductive amination of glycans by streamlined glycan extraction, derivatization, and purification on nonporous graphitized carbon sorbents.…”