Site-Specific N- and O-Glycosylation Analysis of Human Plasma Fibronectin

Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans might mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosite sites were localized by the 18O-labeling method. O-glycopeptide enrichment and O-glycosite identification were achieved by an enzyme-assisted site-specific extraction method. An RP–LC–MS/MS system functionalized with collision-induced dissociation and stepped normalized collision energy (sNCE)-HCD tandem mass was applied to analyze the glycoforms of fibronectin. A total of 6 N-glycosites and 53 O-glycosites were identified, which were occupied by 38 N-glycoforms and 16 O-glycoforms, respectively. Furthermore, 77.31% of N-glycans were sialylated, and O-glycosylation was dominated by the sialyl-T antigen. These site-specific glycosylation patterns on human fibronectin can facilitate functional analyses of fibronectin and therapeutics development.

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A site-specific map of the human plasma glycome and its age and gender-associated alterations

Scientific Reports ◽

10.1038/s41598-020-73588-x ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Alexander A. Merleev ◽

Dayoung Park ◽

Yixuan Xie ◽

Muchena J. Kailemia ◽

Gege Xu ◽

...

Keyword(s):

Human Plasma ◽

Multiple Reaction Monitoring ◽

Reaction Monitoring ◽

Spectrometry Method ◽

Age And Gender ◽

Site Specific ◽

And Gender ◽

Biomarker Research ◽

A Site ◽

Age Prediction

Abstract Alterations in the human glycome have been associated with cancer and autoimmunity. Thus, constructing a site-specific map of the human glycome for biomarker research and discovery has been a highly sought-after objective. However, due to analytical barriers, comprehensive site-specific glycoprofiling is difficult to perform. To develop a platform to detect easily quantifiable, site-specific, disease-associated glycan alterations for clinical applications, we have adapted the multiple reaction monitoring mass spectrometry method for use in glycan biomarker research. The adaptations allow for highly precise site-specific glycan monitoring with minimum sample prep. Using this technique, we successfully mapped out the relative abundances of the most common 159 glycopeptides in the plasma of 97 healthy volunteers. This plasma glycome map revealed 796 significant (FDR < 0.05) site-specific inter-protein and intra-protein glycan associations, of which the vast majority were previously unknown. Since age and gender are relevant covariants in biomarker research, these variables were also characterized. 13 glycopeptides were found to be associated with gender and 41 to be associated with age. Using just five age-associated glycopeptides, a highly accurate age prediction model was constructed and validated (r2 = 0.62 ± 0.12). The human plasma site-specific glycan map described herein has utility in applications ranging from glycan biomarker research and discovery to the development of novel glycan-altering interventions.

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Faculty Opinions recommendation of A mutation in the Mod subunit of EcoP15I restriction enzyme converts the DNA methyltransferase to a site-specific endonuclease.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1074805.527727 ◽

2007 ◽

Author(s):

Narayanaswamy Srinivasan

Keyword(s):

Restriction Enzyme ◽

Dna Methyltransferase ◽

Site Specific ◽

A Site

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Faculty Opinions recommendation of A site-specific, multiplexed kinase activity assay using stable-isotope dilution and high-resolution mass spectrometry.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1161908.623533 ◽

2009 ◽

Author(s):

Jonathan Chernoff

Keyword(s):

Mass Spectrometry ◽

High Resolution ◽

Kinase Activity ◽

High Resolution Mass Spectrometry ◽

Activity Assay ◽

Site Specific ◽

Stable Isotope Dilution ◽

A Site ◽

Kinase Activity Assay ◽

Resolution Mass

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DECADAL SUMMER LAKE WATER TEMPERATURE VARIABILITY IN SOUTHERN GREENLAND OF THE LAST 2 MILLENNIA: DEVELOPMENT AND APPLICATION OF A SITE-SPECIFIC BRGDGT CALIBRATION

10.1130/abs/2020am-355583 ◽

2020 ◽

Author(s):

Boyang Zhao ◽

◽

Isla S. Castañeda ◽

Raymond S. Bradley ◽

Jeff Salacup ◽

...

Keyword(s):

Water Temperature ◽

Lake Water ◽

Temperature Variability ◽

Site Specific ◽

A Site

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Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

Molecular and Cellular Biology ◽

10.1128/mcb.9.4.1507 ◽

1989 ◽

Vol 9 (4) ◽

pp. 1507-1512 ◽

Cited By ~ 22

Author(s):

H Zhu ◽

H Conrad-Webb ◽

X S Liao ◽

P S Perlman ◽

R A Butow

Keyword(s):

Genetic Analysis ◽

Rna Processing ◽

Fold Increase ◽

Functional Expression ◽

Rrna Gene ◽

Yeast Mitochondria ◽

Wild Type ◽

Site Specific ◽

Var1 Gene ◽

A Site

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

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