Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Elsevier Science full text link Elsevier Science Free PMC article
Full text links

Actions

.2013 Apr;12(4):921-31.
doi: 10.1074/mcp.M113.028878. Epub 2013 Mar 1.

Selected reaction monitoring to differentiate and relatively quantitate isomers of sulfated and unsulfated core 1 O-glycans from salivary MUC7 protein in rheumatoid arthritis

Affiliations

Selected reaction monitoring to differentiate and relatively quantitate isomers of sulfated and unsulfated core 1 O-glycans from salivary MUC7 protein in rheumatoid arthritis

Sarah A Flowers et al. Mol Cell Proteomics.2013 Apr.

Abstract

Rheumatoid arthritis is a common and debilitating systemic inflammatory condition affecting up to 1% of the world's population. This study aimed to investigate the immunological significance of O-glycans in chronic arthritis at a local and systemic level. O-Glycans released from synovial glycoproteins during acute and chronic arthritic conditions were compared and immune-reactive glycans identified. The sulfated core 1 O-glycan (Galβ1-3GalNAcol) was immune reactive, showing a different isomeric profile in the two conditions. From acute reactive arthritis, three isomers could be sequenced, but in patients with chronic rheumatoid arthritis, only a single 3-Gal sulfate-linked isomer could be identified. The systemic significance of this glycan epitope was investigated using the salivary mucin MUC7 in patients with rheumatoid arthritis and normal controls. To analyze this low abundance glycan, a selected reaction monitoring (SRM) method was developed to differentiate and relatively quantitate the core 1 O-glycan and the sulfated core 1 O-glycan Gal- and GalNAc-linked isomers. The acquisition of highly sensitive full scan linear ion trap MS/MS spectra in addition to quantitative SRM data allowed the 3- and 6-linked Gal isomers to be differentiated. The method was used to relatively quantitate the core 1 glycans from MUC7 to identify any systemic changes in this carbohydrate epitope. A statistically significant increase in sulfation was identified in salivary MUC7 from rheumatoid arthritis patients. This suggests a potential role for this epitope in chronic inflammation. This study was able to develop an SRM approach to specifically identify and relatively quantitate sulfated core 1 isomers and the unsulfated structure. The expansion of this method may afford an avenue for the high throughput investigation of O-glycans.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Extracted ion chromatograph of the sulfated core 1 isomers atm/z 464.1 was from theO-glycans released from synovial acidic glycoproteins from an ReA patient (A) and RA patient (D). Three isomers were identified in the ReA patient, one GalNAc-linked (RT 15.4 min) and two Gal-linked (RTs 16.6 and 17.2 min). A single Gal-linked isomer was identified in the RA patient (RT 15.6 min). The MS/MS (using a linear ion trap) from the two Gal-linked isomers from the ReA patient (A) are shown inB andC. The 3-linked sulfated isomer is shown (B) with the site-indicative cross-ring fragments and the 6-linked inC. The MS/MS (using a linear ion trap) of the Gal-linked isomer from the RA patient (D) is shown inE with the cross-ring fragments indicating a 3-linked sulfate.Yellow square represents GalNAc;yellow circle is Gal, andS indicates sulfate.
Fig. 2.
Fig. 2.
Comparison of Alcian blue-stained PVDF membranes after SDS-AgPAGE separation and base peak chromatographs from theO-glycans, which were then released by reductive β-elimination of an enriched SF sample from a ReA patient (A andB) and salivary MUC7 from a control sample (C andD). Core 1 structures are indicated on the base peak with [M − H] ions for the core 1O-glycan ofm/z 384.1, sulfated core 1 of m/z 464.1, sialylated core 1 ofm/z 675.2, and disialylated core 1 ofm/z 966.3. The ReA sample shows a major band (A), and whenO-glycans were released, the sample showed an abundance of core 1 structures, with the lower abundance sulfated core 1 structure inlaid (B). The saliva sample shows a higher molecular weight MUC5B and lower molecular weight MUC7 band (C).O-Glycans were released, and the base peak is shown inD. The base peak is more complex than the SF (B) with a single sulfated core 1 peak.
Fig. 3.
Fig. 3.
A, MS/MS of the unsulfated core 1O-glycan with an [M − H] ion atm/z 384.1 fragmented in the QTRAP® ESI-triple quadrupole linear ion trap hybrid mass spectrometer.B, relative intensities of the 10 most intense fragments were collected for 50 MS/MS scans at the standard CE of −35 eV. A boxplot presents these data. A large variation was observed in the relative intensities for the fragments shown by the broad boxes indicating the median and lower first and upper third quartiles. The large ranges for the fragments are shown by the maximum and minimum.
Fig. 4.
Fig. 4.
MS/MS fragmentation of the three possible sulfated isomers with [M − H] ofm/z 464.1 in the QTRAP® ESI triple-quadrupole linear ion trap hybrid mass spectrometer used for SRM analysis. The glycosidic fragments used in SRM transitions are indicated. The GalNAc-linked sulfate isomer is shown inA and the two possible Gal-linked isomers, the 3-linked Gal isomer (B) showing site indicative cross-ring fragments (inlaid) and the 6-linked Gal isomer (C).Yellow square represents GalNAc;yellow circle is Gal, andS indicates sulfate.
Fig. 5.
Fig. 5.
SRM trace of the final SRM method using the ReA SF and PGM releasedO-glycans that were used for SRM method development. The three transitions are observed. The 384/101 transition (indicating the unsulfated core 1 structure) is shown inblue. The 464/302 transition (indicating the GalNAc-linked sulfate isomer) is shown ingreen. The 464/241 transition (indicating the Gal-linked sulfate isomers) is displayed inred showing two peaks for the two isomers. The faster gradient for the SRM as described under “Experimental Procedures” was used for the experiments.
Fig. 6.
Fig. 6.
O-Glycans were released by reductive β-elimination from the salivary MUC7 of 10 control and 11 RA patients after separation of whole saliva by SDS-AgPAGE. SRM traces of the control (A) and RA patients (B) show the 384/101 transition (indicating the unsulfated core 1 structure) is shown inblue, and 464/241 transition (indicating the Gal-linked isomer) is shown inred. The single Gal-linked isomer was shown to be the 3-linked sulfate in all samples. The SRM method was used to relatively quantitate the proportion of sulfated core 1O-glycan compared with the unsulfated core 1O-glycan. The data are shown graphically (C) as the percentage of sulfated and unsulfated core 1O-glycan in each sample. A two-tailedt test showed a statistically significant difference (p value 0.0055), with an increase in sulfation in the RA patients. The faster gradient for the SRM as described under “Experimental Procedures” was used for the experiments.
See this image and copyright information in PMC

References

    1. Hennet T. (2012) Diseases of glycosylation beyond classical congenital disorders of glycosylation. Biochim. Biophys. Acta 1820, 1306–1317 - PubMed
    1. Durand G., Seta N. (2000) Protein glycosylation and diseases: blood and urinary oligosaccharides as markers for diagnosis and therapeutic monitoring. Clin. Chem. 46, 795–805 - PubMed
    1. Hart G. W., Copeland R. J. (2010) Glycomics hits the big time. Cell 143, 672–676 - PMC - PubMed
    1. McGuckin M. A., Lindén S. K., Sutton P., Florin T. H. (2011) Mucin dynamics and enteric pathogens. Nat. Rev. Microbiol. 9, 265–278 - PubMed
    1. Hollingsworth M. A., Swanson B. J. (2004) Mucins in cancer: protection and control of the cell surface. Nat. Rev. Cancer 4, 45–60 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources

Full text links
Elsevier Science full text link Elsevier Science Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2026 Movatter.jp