Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2).

Martina Pirro,Arnoud H De Ru,Katarina Madunić,Rayman T N Tjokrodirijo,Peter A Van Veelen,Paul J Hensbergen,Manfred Wuhrer,George M C Janssen,Yassene Mohammed

doi:10.3390/ijms22105369

Abstract

Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets.

Highlights

Nowadays, large-scale mass spectrometry (MS)-based proteomics is widely used in biomedical research, to investigate overall changes in protein abundance, for example by isotopic labeling approaches (e.g., SILAC, dimethyl labeling, and tandem mass tagging (TMT), and to analyze post-translational modifications [1]
Differentiation of Glycopeptides Based on the Relative Abundance of Oxonium Ions
To speed up the database searches for glycopeptides, we have recently developed a tool that extracts MS/MS spectra based on the presence of the HexNAc oxonium ion at m/z

Summary

Introduction

Large-scale mass spectrometry (MS)-based proteomics is widely used in biomedical research, to investigate overall changes in protein abundance, for example by isotopic labeling approaches (e.g., SILAC, dimethyl labeling, and tandem mass tagging (TMT), and to analyze post-translational modifications [1]. In order to reduce the complexity of the sample, enrichment of modified peptides/proteins is often applied prior to the downstream mass spectrometry analysis [2]. Unique characteristics of MS/MS fragmentation patterns, such as the presence of diagnostic ions, have been exploited to aid in the analysis and confident identification of peptides in general, and post-translationally modified peptides in particular [3,4]. MS/MS analysis of intact glycopeptides can aid the identification of glycan structures in a protein specific manner, but multiple fragmentation techniques are usually required to determine site-specific (changes in) protein glycosylation. Their full characterization is far from trivial and several challenges for the assignment of glycopeptide MS/MS spectra are, amongst others, the dominance of glycosidic bond cleavages over peptide bond cleavages with most commonly applied fragmentation techniques, the complexity of potential glycan structures that can be present, long search times, especially for O-glycopeptides, and discrimination between isobaric structures. To simplify the glycan structures, glycoengineering of cellular systems [11], and glycosidases [12], have been used

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Results