Dataset Information

Ion Mobility-Tandem Mass Spectrometry of Mucin-type O-Glycans

ABSTRACT: The dense O-glycosylation of mucins plays an important role in the defensive properties of the mucus hydrogel. Aberrant glycosylation is often correlated with inflammation and pathology such as COPD, cancer, and Crohn’s disease. The inherent complexity of glycans and the diversity in the O-core structure constitute fundamental challenges for the analysis of mucin-type O-glycans. Due to coexistence of multiple isomers, multidimensional workflows such as LC-MS are required. To separate the highly polar carbohydrates, porous graphitized carbon is often used as a stationary phase. However, LC-MS workflows are time-consuming and lack reproducibility. Here we present a rapid alternative for separating and identifying O-glycans released from mucins based on trapped ion mobility mass spectrometry. Compared to established LC-MS, the acquisition time is reduced from an hour to two minutes. To test the validity, the developed workflow was applied to sputum samples from cystic fibrosis patients to map O-glycosylation features associated with disease.

INSTRUMENT(S): timsTOF Pro, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human) Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Sputum

DISEASE(S): Cystic Fibrosis

SUBMITTER: Marieluise Kirchner

LAB HEAD: Kevin Pagel (Glycomics), Philipp Mertins (Proteomics)

PROVIDER: PXD050530 | Pride | 2024-03-19

REPOSITORIES: Pride

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Publications

Ion mobility-tandem mass spectrometry of mucin-type O-glycans.

Bechtella Leïla L Chunsheng Jin J Fentker Kerstin K Ertürk Güney R GR Safferthal Marc M Polewski Łukasz Ł Götze Michael M Graeber Simon Y SY Vos Gaël M GM Struwe Weston B WB Mall Marcus A MA Mertins Philipp P Karlsson Niclas G NG Pagel Kevin K

Nature communications 20240323 1

The dense O-glycosylation of mucins plays an important role in the defensive properties of the mucus hydrogel. Aberrant glycosylation is often correlated with inflammation and pathology such as COPD, cancer, and Crohn's disease. The inherent complexity of glycans and the diversity in the O-core structure constitute fundamental challenges for the analysis of mucin-type O-glycans. Due to coexistence of multiple isomers, multidimensional workflows such as LC-MS are required. To separate the highly ...[more]

PMID: 38521783

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Project description:The phylum Bacteroidetes is a major component of the human gut microbiota which has a broad impact on the development and physiology of its host, and a potential role in a wide range of disease syndromes1-3. The predominance of Bacteroidetes and the genus Bacteroides in the distal gut is due in large part to the expansion of paralogous gene clusters, termed Polysaccharide Utilization Loci (PULs), dedicated to the uptake and catabolism of host derived and dietary polysaccharides4,5. It is generally thought that the diversity of PULs is key to Bacteroides successful competition for nutrients in the gut environment6. The nutritive value of the available polysaccharides varies greatly and thus their utilization is hierarchical and strictly controlled. A typical PUL includes regulatory genes that control expression in response to the presence of specific glycan substrates. However the existence of additional regulatory mechanisms has been predicted to explain phenomena such as the hierarchical control, catabolite repression, and the fine tuning of gene expression to match catabolic activity7-9. Using Bacteroides fragilis as a model organism, this report describes a previously unknown layer of regulatory control in which cis-encoded antisense small RNAs (sRNA) act as repressors of the PULsâ catabolic genes. Nearly 30% of B. fragilis PULs are subject to this type of sRNA control and these PULs tend to be more closely linked to the utilization of host-derived glycans than dietary polysaccharides. The findings described here indicate the presence of a global control mechanism that underlies the known regulatory circuits which modulate PUL expression in response to substrate availability, and hence provide novel insight into regulation of the gut Bacteroidetes physiology. This is a 4 chip study with 8 technical replicates on each chip. This was an in vitro, exploratory study to determine if mutation or overexpression of a sRNA associated with the Don locus would affect gene expression. In vitro cultures were grown in defined media with mucin glycans as the sole carbon source. The two chips representing growth of the wild type strain (638R) on mucin glycans were also used in a related study GSE53883 (GSM1303101 and GSM1303102).

Project description:Alterations in glycosylation are seen in many types of cancer, including colorectal cancer (CRC). CRC is known to display glycosylation alterations. Glycans, the sugar moieties of glycoconjugates, are involved in many important functions relevant to cancer, such as cell signaling and adhesion, and may can be of value as biomarkers. In this study, we have used mass spectrometry to analyze the N-glycan profiles of 35 CRC tissue samples from patients with tumors in the right or left colon and 10 healthy tissue samples from non-CRC patients who underwent operations for other reasons. The tumor samples were divided into groups depending on tumor location (right or left colon) and stage (II or III), while the healthy samples were divided into right or left side of the colon. The levels of neutral and acidic N-glycan compositions and glycan classes were analyzed in a total of ten different groups. Surprisingly, there were no significant differences in glycan levels when all right- and left-sided CRC samples were compared, and few differences (such as in the abundance of the neutral N-glycan H3N5) were seen when the samples were divided according to both location and stage. Multiple significant differences were found in the levels of glycans and glycan classes when stage II and III samples were compared, and these glycans could be of value as candidates for new markers of cancer progression. In order to validate our findings, we analyzed healthy tissue samples from the right and left colon and found no significant differences in the levels of any of the glycans analyzed, confirming that our findings when comparing CRC samples from the right and left colon are not due to normal variations in the levels of glycans between the healthy right and left colon. Additionally, the levels of the acidic glycans H4N3F1P1, H5N4F1P1, and S1H5N4F1 were found to change in a cancer-specific but colon location-nonspecific manner, indicating that CRC affects glycan levels in similar ways, regardless of tumor location.

Dataset Information

Ion Mobility-Tandem Mass Spectrometry of Mucin-type O-Glycans

Publications

Ion mobility-tandem mass spectrometry of mucin-type O-glycans.

Similar Datasets

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