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

Abstract

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 (R 2 > 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).

Oxonium Ion–Guided Optimization of Ion Mobility–Assisted Glycoproteomics on the timsTOF Pro

“…Recently, IM separation mass spectrometry (MS) has emerged as a promising tool for characterizing glycosylated species ( 28 , 29 , 30 , 31 ). Glycopeptides, because of their inherent physical properties, have been shown to typically separate from nonmodified peptides within both drift-tube and traveling-wave IM mass spectrometers ( 32 , 33 ).…”

Determination of Sialic Acid Isomers from Released N-Glycans Using Ion Mobility Spectrometry

“… 15 The method described here is therefore well suited to quantitatively describe the ratio of α2,6- and α2,3-linked Neu5Ac isomers without chromatographic separation. In comparison to the corresponding glycopeptide workflows, 21 , 22 , 31 it is more straightforward to identify sialic acid isomers on the level of released glycans. Sialic acid isomers of complex N -glycans (like hAGP) can be identified and quantified separately for individual N -glycan classes as bi-, tri-, and tetraantennary glycans.…”

Ion Mobility Mass Spectrometry for the Separation and Characterization of Small Molecules

“…Apart from using chiral selectors in the separation process, the common method to increase the difference of CCS is chemical derivatization before separation. , The stereoisomers that are difficult to separate with a CCS difference of less than 2% can be distinguished by combining chemical derivatization with IM-MS. − Amino acids and oligosaccharides are the most difficult to distinguish small molecular stereoisomers, and the most commonly used chemical derivatives are metal cations and halogen anions. ,− For example, McKenna et al used 3-carboxy-5-nitrophenylboronic acid to amplify the structural difference between oligosaccharides and then used the IM-MS method to separate and identify the main products after derivatization. In order to effectively separate and quantify (+)-catechin and (−)-epicatechin, Bian et al complexed the two configurations with cyclodextrin; the difference between CCS expanded from 0.32 Å 2 to 11.75 Å 2 , and the separation degree reached 0.86 in the IM dimension after complexation.…”