Dataset Information

Glyco-seek: Ultrasensitive Detection of Protein-Specific Glycosylation by Proximity Ligation Polymerase Chain Reaction.

ABSTRACT: We report a non-destructive biochemical technique, termed "Glyco-seek", for analysis of O-GlcNAcylated proteins. Glyco-seek combines chemoenzymatic labeling, proximity ligation, and quantitative polymerase chain reaction to detect O-GlcNAcylated proteins with ultrahigh sensitivity. Our glycan-specific assay can be paired with traditional proximity ligation assays to simultaneously determine the change in total protein levels. We show that Glyco-seek detects attomoles of glycoproteins of interest from cell lysates, with sensitivity several orders of magnitude higher than that of current techniques. We used the method to directly assay the O-GlcNAcylation status of a low-abundance transcription factor from cell lysates without need for isolation or enrichment.

SUBMITTER: Robinson PV

PROVIDER: S-EPMC5327792 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Publications

Glyco-seek: Ultrasensitive Detection of Protein-Specific Glycosylation by Proximity Ligation Polymerase Chain Reaction.

Robinson Peter V PV Tsai Cheng-Ting CT de Groot Amber E AE McKechnie Julia L JL Bertozzi Carolyn R CR

Journal of the American Chemical Society 20160817 34

We report a non-destructive biochemical technique, termed "Glyco-seek", for analysis of O-GlcNAcylated proteins. Glyco-seek combines chemoenzymatic labeling, proximity ligation, and quantitative polymerase chain reaction to detect O-GlcNAcylated proteins with ultrahigh sensitivity. Our glycan-specific assay can be paired with traditional proximity ligation assays to simultaneously determine the change in total protein levels. We show that Glyco-seek detects attomoles of glycoproteins of interest ...[more]

PMID: 27454154

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Project description:ImportanceThe diagnostic workup of patients suspected of having vitreoretinal lymphoma (VRL) is primarily based on vitreous fluid analysis, including the recently emerging myeloid differentiation primary response gene 88 (MYD88) mutation analysis. Aqueous humor paracentesis is a relatively less invasive and safer procedure than taking vitreous fluid specimens, and aqueous humor-based MYD88 mutation analysis would provide an additional liquid biopsy tool to diagnose and monitor patients with VRL.ObjectiveTo investigate whether the detection of MYD88 L265P by highly sensitive droplet digital polymerase chain reaction (ddPCR) is feasible in the vitreous fluid and aqueous humor of patients with VRL.Design, setting, and participantsThis cohort study includes aqueous humor and vitreous fluid samples from patients with VRL who were treated at the University Medical Center Utrecht, in Utrecht, the Netherlands, from August 2005 to August 2017. Ocular fluids were randomized and masked before MYD88 L265P analysis, which was performed using an in-house validated ddPCR platform. Patients with uveitis were included as a comparison group.Main outcomes and measuresThe presence of MYD88 L265P mutation detected by ddPCR in AH and VF.ResultsThe study included 96 samples from 63 individuals, including 23 patients with VRL (of whom 10 were female and 13 male, with a mean [SD] age of 72 [7.3] years) and 40 individuals with uveitis (of whom 23 were female and 17 male, with a mean [SD] age of 58 [20.9] years). In 17 of 23 patients with VRL (74%), MYD88 L265P was detected; it was not detected in any of the patients with uveitis. It was detectable in both vitreous fluid and aqueous humor samples. In the paired samples, the mutation was detected in 8 of 9 aqueous humor samples (89%) of the MYD88 L265P-positive vitreous fluid samples. In vitreous fluid, the MYD88 ddPCR test showed a sensitivity of 75% (95% CI, 50%-92%) and a positive predictive value of 100%; in aqueous humor, sensitivity was 67% (95% CI, 42%-92%), and positive predictive value was 100%. Specificity was 100% in both fluids. After treatment, the mutation was no longer detectable in any ocular fluids.Conclusions and relevanceThe high concordance between aqueous humor and vitreous fluid samples suggests that use of the easily accessible aqueous humor is nearly as informative as vitreous fluid in the identification of key somatic mutations in patients with VRL. This approach may provide an additional minimally invasive tool for accurate diagnosis, detection of recurrence, and monitoring of treatment.

Dataset Information

Glyco-seek: Ultrasensitive Detection of Protein-Specific Glycosylation by Proximity Ligation Polymerase Chain Reaction.

Publications

Glyco-seek: Ultrasensitive Detection of Protein-Specific Glycosylation by Proximity Ligation Polymerase Chain Reaction.

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