Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)
Date
2021
Authors
Pirro, Martina
Mohammed, Yassene
de Rue, Arnoud H.
Janssen, George M. C.
Tjokrodirijo, Rayman T. N.
Madunić, Katarina
Wuhrer, Manfred
van Veelen, Peter A.
Hensbergen, Paul J.
Journal Title
Journal ISSN
Volume Title
Publisher
International Journal of Molecular Sciences
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.
Description
Keywords
glycoproteomics, TMT labeling, oxonium ion, LC-MS/MS, O-glycosylation, colorectal cancer, AGR2
Citation
Pirro, M., Mohammed, Y., de Rue, A. H., Janssen, G. M. C., Tjokrodirijo, R. T. N., Madunić, K., … Hensbergen, P. J. (2021). Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2). International Journal of Molecular Sciences, 22(10), 1-15. https://doi.org/10.3390/ijms22105369.