Abstract: In an in vitro method of determining the modification degree of O-GlcNAc in a biological sample, a first sample is treated with B3GALNT2 to incorporate a GalNAz into closed O-GlcNAc sites in the sample, labeled using click chemistry to form labeled closed O-GlcNAc sites, and the labeled closed O-GlcNAc sites in the first sample are detected (corresponding to the number of closed O-GlcNAc sites in the biological sample). A second, duplicate sample is treated with OGT to O-GlcNAcylate open O-GlcNAc sites in the sample to convert the open O-GlcNAc sites to closed O-GlcNAc sites, and treated with B3GALNT2 to incorporate GalNAz into the closed O-GlcNAc sites. The second sample is labeled using click chemistry to form labeled closed O-GlcNAc sites (corresponding to the total number of O-GlcNAc sites in the biological sample). The number of closed O-GlcNAc sites and total O-GlcNAc sites in the biological sample are compared to determine the modification degree of O-GlcNAc.

Abstract: A method for assaying endoglycosidase activity includes providing a proteoglycan having a glycosaminoglycan chain with a non-reducing end; treating the proteoglycan with a glycosyltransferase to incorporate a carbohydrate into the non-reducing end of the glycosaminoglycan chain, wherein the carbohydrate includes a click chemistry moiety; adding a label to the proteoglycan, wherein the label includes a click chemistry moiety that reacts to the click chemistry moiety of the carbohydrate such that the label attaches to the carbohydrate to form a labeled proteoglycan; immobilizing the labeled proteoglycan on a multi-well plate, wherein the multi-well plate includes a specific anti-proteoglycan antibody for binding the labeled proteoglycan; treating the labeled proteoglycan with an endoglycosidase specific to the glycosaminoglycan chain; and detecting the labeled proteoglycan.

Abstract: An in vitro method of imaging a cancer antigen includes providing a cell sample including a cancer antigen selected from the group consisting of a Tn antigen, a T antigen, a sialylated-Tn antigen, and a sialylated-T antigen, treating the sample with a glycosyltransferase to incorporate a carbohydrate with a click chemistry moiety into the cancer antigen, adding a label to the sample that includes a click chemistry moiety that reacts to the click chemistry moiety of the carbohydrate such that the label attaches to the carbohydrate to form a labeled cancer antigen, and imaging the sample with a camera.

Abstract: A method for assaying endoglycosidase activity includes providing a proteoglycan having a glycosaminoglycan chain with a non-reducing end; treating the proteoglycan with a glycosyltransferase to incorporate a carbohydrate into the non-reducing end of the glycosaminoglycan chain, wherein the carbohydrate includes a click chemistry moiety; adding a label to the proteoglycan, wherein the label includes a click chemistry moiety that reacts to the click chemistry moiety of the carbohydrate such that the label attaches to the carbohydrate to form a labeled proteoglycan; immobilizing the labeled proteoglycan on a multi-well plate, wherein the multi-well plate includes a specific anti-proteoglycan antibody for binding the labeled proteoglycan; treating the labeled proteoglycan with an endoglycosidase specific to the glycosaminoglycan chain; and detecting the labeled proteoglycan.

Abstract: A kit for detecting or measuring glycosyltransferase activity including a first reagent comprising a phosphatase and a second reagent comprising a free phosphate detector, and method of detecting and measuring glycosyltransferase activity. A sugar donor, an acceptor substrate, a glycosyltransferase enzyme and a phosphatase are combined and the amount of free phosphate present in the product is measured and used to calculate the activity of the glycosyltransferase enzyme.

Abstract: Methods and kits for detecting the presence of GlcNAc modification of a peptide or protein. The steps of detection may include combining the peptide or protein with PAP35S and a sulfotransferase to produce a reaction product including 35S labeled GlcNAc modified protein or peptide, removing background labeling with a glycosidase, separating the 35S labeled GlcNAc modified protein or peptide from PAP35S, free S-35 sulfate and labeled oligosaccharides if present, and detecting isotopic emissions from the 35S labeled GlcNAc modified peptide or protein. The detection may further include, prior to detection, the step of combining the peptide or protein with a GlcNAc transferase and UDP-GlcNAc under appropriate conditions for the GlcNAc transferase to transfer a GlcNAc to the peptide or protein.

Abstract: The disclosed methods involve deglycosylating a target molecule to remove target carbohydrates to create vacant glycosylation acceptor sites and then using glycosyltransferases to incorporate replacement carbohydrates into those sites. The methods also involve using glycosytransferases to incorporate new carbohydrates into vacant glycosylation acceptor sites without having to perform in vitro deglycosylation. The replacement or new carbohydrates include a click chemistry moiety that reacts to a click chemistry moiety on a label.

Abstract: The disclosure relates to in vitro methods of detecting presence or absence of a target carbohydrate on a glycoprotein. The disclosure also relates to in vitro methods of detecting presence or absence of a glycan epitope on a glycoprotein.

Abstract: A method of detecting sulfotransferase activity including conducting a first reaction comprising, measuring free phosphate produced by the first reaction, and comparing the measured free phosphate to a free phosphate standard curve or equation or to a free phosphate level obtained in a separate reaction. The first reaction includes combining the sulfotransferase, Golgi-resident PAP-phosphatase (gPAPP), a 5?-nucleotidase, a substrate of the sulfotransferase, and 3?-phosphoadenosine-5?-phosphosulfate (PAPS) under conditions to produce 3?-phosphoadenosine-5?-phosphate (PAP).

Abstract: Methods and kits for detecting the presence of GlcNAc modification of a peptide or protein. The steps of detection may include combining the peptide or protein with PAP35S and a sulfotransferase to produce a reaction product including 35S labeled GlcNAc modified protein or peptide, removing background labeling with a glycosidase, separating the 35S labeled GlcNAc modified protein or peptide from PAP35S, free S-35 sulfate and labeled oligosaccharides if present, and detecting isotopic emissions from the 35S labeled GlcNAc modified peptide or protein. The detection may further include, prior to detection, the step of combining the peptide or protein with a GlcNAc transferase and UDP-GlcNAc under appropriate conditions for the GlcNAc transferase to transfer a GlcNAc to the peptide or protein.

Abstract: A method of detecting sulfotransferase activity including conducting a first reaction comprising, measuring free phosphate produced by the first reaction, and comparing the measured free phosphate to a free phosphate standard curve or equation or to a free phosphate level obtained in a separate reaction. The first reaction includes combining the sulfotransferase, Golgi-resident PAP-phosphatase (gPAPP), a 5?-nucleotidase, a substrate of the sulfotransferase, and 3?-phosphoadenosine-5?-phosphosulfate (PAPS) under conditions to produce 3?-phosphoadenosine-5?-phosphate (PAP).

Abstract: Assays and methods for detecting and/or quantifying activity of a test sulfotransferase comprising by releasing a free phosphate from the PAP produced by the sulfotransferase reaction and detecting the free phosphate. The assay can includes gPAPP, a free phosphate detector and an optional control sulfotransferase. The method includes combining a test sulfotransferase with the substrate of the sulfotransferase, PAPS and gPAPP and detecting the free phosphate. The level of free phosphate directly correlates to the activity of the sulfotransferase. The assay and methods can be used to screen agents for their effect upon sulfotransferase activity.