Identification of salivary N-glycoproteins and measurement of glycosylation site occupancy by boronate glycoprotein enrichment and liquid chromatography/electrospray ionization tandem mass spectrometry

Xu, Ying, Bailey, Ulla-Maja, Punyadeera, Chamindie and Schulz, Benjamin L. (2014) Identification of salivary N-glycoproteins and measurement of glycosylation site occupancy by boronate glycoprotein enrichment and liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Communications In Mass Spectrometry, 28 5: 471-482. doi:10.1002/rcm.6806


Author Xu, Ying
Bailey, Ulla-Maja
Punyadeera, Chamindie
Schulz, Benjamin L.
Title Identification of salivary N-glycoproteins and measurement of glycosylation site occupancy by boronate glycoprotein enrichment and liquid chromatography/electrospray ionization tandem mass spectrometry
Journal name Rapid Communications In Mass Spectrometry   Check publisher's open access policy
ISSN 0951-4198
1097-0231
Publication date 2014-03-15
Sub-type Article (original research)
DOI 10.1002/rcm.6806
Volume 28
Issue 5
Start page 471
End page 482
Total pages 12
Place of publication Chichester, West Sussex, United Kingdom
Publisher John Wiley & Sons
Collection year 2015
Language eng
Formatted abstract
RATIONALE Diseases including cancer and congenital disorders of glycosylation have been associated with changes in the site-specific extent of protein glycosylation. Saliva can be non-invasively sampled and is rich in glycoproteins, giving it the potential to be a useful biofluid for the discovery and detection of disease biomarkers associated with changes in glycosylation.

METHODS Saliva was collected from healthy individuals and glycoproteins were enriched using phenylboronic acid based glycoprotein enrichment resin. Proteins were deglycosylated with peptide-N-glycosidase F and digested with AspN or trypsin. Desalted peptides and deglycosylated peptides were separated by reversed-phase liquid chromatography and detected with on-line electrospray ionization quadrupole-time-of-flight mass spectrometry using a 5600 TripleTof instrument. Site-specific glycosylation occupancy was semi-quantitatively determined from the abundance of deglycosylated and nonglycosylated versions of each given peptide.

RESULTS Glycoprotein enrichment identified 67 independent glycosylation sites from 24 unique proteins, a 3.9-fold increase in the number of glycosylation sites identified. Enrichment of glycoproteins rather than glycopeptides allowed detection of both deglycosylated and nonglycosylated versions of each peptide, and thereby robust measurement of site-specific occupancy at 21 asparagines. Healthy individuals showed limited biological variability in occupancy, with partially modified sites having characteristics consistent with inefficient glycosylation by oligosaccharyltransferase. Inclusion of negative controls without enzymatic deglycosylation controlled for spontaneous chemical deamidation, and identified asparagines previously incorrectly annotated as glycosylated.

CONCLUSIONS We developed a sample preparation and mass spectrometry detection strategy for rapid and efficient measurement of site-specific glycosylation occupancy on diverse salivary glycoproteins suitable for biomarker discovery and detection of changes in glycosylation occupancy in human disease.
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2015 Collection
School of Chemistry and Molecular Biosciences
UQ Diamantina Institute Publications
 
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Created: Fri, 14 Feb 2014, 09:58:01 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences