title: Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti(4+)-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.
authors: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China; Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032, PR China.
authors: Ying
authors: Y
authors: Zhang
authors: Department of Chemistry, Fudan University, Shanghai 200032, PR China.
authors: Ye
authors: Y
authors: Peng
authors: Department of Chemistry, Fudan University, Shanghai 200032, PR China.
authors: Zhichao
authors: Z
authors: Bin
authors: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China.
authors: Huijie
authors: H
authors: Wang
authors: Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China; Department of Chemistry, Fudan University, Shanghai 200032, PR China; Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032, PR China. Electronic address: luhaojie@fudan.edu.cn.
authors: Haojie
authors: H
authors: Lu
mesh_terms: D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D015394:Molecular Structure; D010710:Phosphates / Q000737:chemistry*; D011134:Polysaccharides / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D052616:Solid Phase Extraction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014025:Titanium / Q000737:chemistry*
chemical_list: D006023:Glycoproteins; D010710:Phosphates; D011134:Polysaccharides; D014025:Titanium
keywords: Derivatization; Enrichment; MALDI-MS; N-glycan; Ti(4+)-SPE