Quantification by isobaric labeling (QUIBL) for the comparative glycomic study of O-linked glycans

Quantification by isobaric labeling (QUIBL) has recently been described as a tool for the relative quantification of N-linked glycans in glycomic studies. In this paper we expand the application of QUIBL to the relative quantification of O-linked glycans. Fetuin, which contains two O-linked glycans, NeuAc-Hex-HexNAc and NeuAc2-Hex-HexNAc, was used to validate this study. The glycans were released by β-elimination and permethylated with the isobaric labeling reagents, 13CH3I and 12CH2DI. The exact mass difference between the isobaric labels is minimal (0.00292 Da), but since glycans contain multiple permethylation sites, the mass difference can be resolved with a high resolution mass spectrometer, such as an FTMS. Quantitative data were obtained by comparing the signal intensities from various mixtures of isobarically labeled O-linked glycans of fetuin. Five different ratios of 13CH3 to 12CH2D ranging from 10:1 to 1:10 were analyzed for linearity and reproducibility of the QUIBL method on the standard glycan. QUIBL was also evaluated using porcine mucin, a sample with a larger variety of more complex O-linked glycans. These results indicate that QUIBL allows for the relative quantification of O-linked glycans over a linear dynamic range of at least two orders of magnitude, validating QUIBL as an applicable quantitative glycomics approach for both N-linked and O-linked glycans.