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

• A highly specific N-glycans purification method based on phosphate derivatization combined with Ti4+-SPE was developed.
• The new method showed high derivatization efficiency (almost 100%), excellent selectivity (1:100), and good enriching recovery (90%).
• Better selectivity and good enrichment recovery were also shown by comparing with commercial enrichment/clean-up protocols.

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 Ti4+-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 Ti4+-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.

A selective enrichment method for the N-glycome is reported. N-glycans were chemically labeled with a phosphate derivatization reagent (AMS), then the phospho-containing glycans were enriched using Ti4+-microspheres.Figure optionsDownload as PowerPoint slide