Relative Quantification of N-Glycopeptide Sialic Acid Linkage Isomers by Ion Mobility Mass Spectrometry.

Sialic acids decorate the surface of glycoproteins and play important roles in a variety of pathological processes. Although the mass spectrometry (MS) based linkage-specific analysis of sialylated N-glycopeptide is developing rapidly, quantitative analysis of these isomers still remains a challenge. Herein, we reported a novel quantitative strategy that can unambiguously identify and relatively quantify linkage-specific N-glycopeptides using ion mobility mass spectrometry (IM-MS). Without the assistance of derivatization, this method can relatively quantify sialic acid isomers of intact glycopeptides by using their characteristic fragment ions in IM-MS. Moreover, good linearity (R2 > 0.99) of relative quantification within a dynamic range of 2 orders of magnitude and high reproducibility (coefficient of variation (CV) < 10%, n = 3) were demonstrated. Finally, our results illustrated the aberrant sialylation of haptoglobin (Hp) in hepatocellular carcinoma (HCC), where the ratios of α2,3 to α2,6 sialylation of seven N-glycopeptides were found to be significantly altered (p < 0.01) in HCC individuals (n = 27) compared with healthy controls (n = 27).