A novel, simplified strategy of relative quantificationN-glycan: Quantitative glycomics using electrospray ionization mass spectrometry through the stable isotopic labeling by transglycosylation reaction of mutant enzyme Endo-M-N175Q

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Abstract

The lack of a highly sensitive and simple method for the quantitative analysis of glycan has impeded the exploration of protein glycosylation patterns (glycomics), evaluation of antibody drug stability, and screening of disease glycan biomarkers. In this study, we describe a novel and simplified quantitative glycomics strategy. Quantitation by mutant enzyme reaction stable isotope labeling (QMERSIL) to label the N-glycans with either a nondeuterated (d0-) or deuterated (d8-) 4-(2,4-Dinitro-5-piperazin-1-yl-phenyl)-1,1-dimethyl-piperazin-1-ium (MPDPZ)-Boc-asparaginyl-N-acetyl-d-glucosamine (Boc-Asn-GlcNAc) acceptor of a positive charge structure through the glycosynthase (Endo-M-N175Q) transglycosylation reaction with mass spectrometry facilitates comparative glycomics. The sialylglycopeptide (SGP) of the complex type was used to demonstrate that QMERSIL facilitates the relative quantitation over a linear dynamic range (up to d0/d8 = 0.02:20) of 3 orders of magnitude. The area ratios of the N-glycan peaks from the QMERSIL method showed a good linearity (d0/d8, R2 = 0.9999; d8/d0, R2 = 0.9978). The reproducibility and accuracy assay precisions were all less than 6.12%, and the mean recoveries (%) of SGP spiked in the human plasma were 97.34%. Moreover, the QMERSIL using LC–MS/MS was evaluated with various molar ratios (1:1, 1:5, 5:1) of d0(d8)- MPDPZ-Boc-Asn-GlcNAc-labeled glycans from ribonuclease B, bovine fetuin, and ovalbumin. The ratios of the relative intensity between the isotopically MPDPZ-Boc-Asn-GlcNAc labeled N-glycans were almost equal a close to the theoretical values (1:1, 1:5, 5:1). Finally, this method was used for the relative quantitative comparison of the N-Linked oligosaccharides in human plasma.

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