Orela O-Linked Glycan Release Kit
The kit includes reagents and materials for O-linked deglycosylation of up to 12 glycoprotein samples analysed in parallel or two sets of 6 samples, typically up to 1 mg of glycoprotein per sample. This method of O-glycan release demonstrates significant reduction in peeling and reduces the number of sample handling steps prior to release.
– Designed for easy, O-linked deglycosylation by QC labs.
– Straightforward and simple method.
– Released glycans have free reducing terminii suitable for fluorescent labelling prior to analysis by HPLC and MS.
– Fluorescent labelling allows relative quantitation of glycan species by HPLC.
– Faster and lower cost than hydrazinolysis (LL-HYDRAZ-A2).
– No special handling techniques.
The Orela Reaction
The Orela reagent reacts at the link between the glycan and peptide backbone to liberate glycans as the glycosylamine. The acid-labile glycosylamine derivative is hydrolysed to produce the free glycan.
Orela Kit Components
|LL-ORELAREAGENT-0||Orela Release Reagent||2 x 3ml|
|LL-ACETIC-50P-01||Acetic Acid Solution||2 x 3ml|
|LC-CEX-H-01||LudgerClean CEX-H cartridges||2 x 6 cartridges|
|LL-COLLV-01||Collection vials with PTFE lined caps||2 x 6 vials|
|LL-REACT-01||Reaction vials with PTFE lined caps||2 x 6 vials|
Outline of the Orela Protocol
Prepare the glycoconjugate
Prepare the glycoprotein or glycopeptide samples by removing contaminants such as salts, detergents and dyes that could interfere with the labeling procedure.
Dry the glycans
Place the samples in reaction vials and dry down.
Add release reagent to glycoconjugates
Add Orela release reagent to each sample.
Incubate the samples to allow the release reaction to progress.
Add acetic acid solution and incubate at 4C to convert glycosylamines into non-reduced glycans
Remove the peptide or protein using a cation exchange column
Store or Analyse released glycans
The released glycans are now ready for analysis
O-linked Deglycosylation References
Kozak RP, Royle L, Gardner RA, Bondt A, Fernandes DL, Wuhrer M Improved nonreductive O-glycan release by hydrazinolysis with ethylenediaminetetraacetic acid addition. Anal Biochem. May 15;453:29-37 (2014).
Kozak RP, Royle L, Gardner RA, Fernandes DL, Wuhrer M. Suppression of peeling during the release of O-glycans by hydrazinolysis. Anal Biochem. 2012 Apr 1;423(1):119-28.
Sojar, H. T. and O.P. Bahl. A chemical method for the deglycosylation of proteins. Arch Biochem Biophys 259:52-57 (1987).
Turyan I, Hronowski X, Sosic Z, Lyubarskaya Y. Comparison of two approaches for quantitative O-linked glycan analysis used in characterization of recombinant proteins. Anal Biochem. Feb 1;446:28-36 (2014).