Fast and efficient online release of N-glycans from glycoproteins facilitating liquid chromatography-tandem mass spectrometry glycomic profiling

Yazen Jmeian, Loubna A. Hammad, Yehia Mechref

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

A novel online enzyme reactor incorporating peptide-N-glycosidase F (PNGase F) on a monolithic polymer support has been developed to allow the rapid simultaneous release of both neutral and acidic N-linked glycans from glycoproteins. The PNGase F monolithic reactor was fabricated in a fused silica using glycidyl methacrylate-co-ethylene dimethacrylate polymer. The reactor was coupled to a C8 trap and a porous graphitic carbon (PGC) HPLC-chip. This arrangement was interfaced to an ion trap mass spectrometer for liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The performance of the PNGase F reactor was optimized using the MS signal for the disialylated biantennary N-glycan derived from fetuin. Optimum conditions for glycan release were attained at room temperature using a loading flow rate of 2 μL/min and a reaction time of 6 min. The loading capacity of the reactor was determined to be around 2 pmol of glycoprotein. The online digestion and MS characterization experiments resulted in sensitivities as high as 100 fmol of glycoprotein and 0.1 μL of human blood serum. The enzyme reactor activity was also shown to remain stable after 1 month of continuous use. Both small and large glycoproteins as well as glycoproteins containing high-mannose glycans, fucolsylated glycans, sialylated glycans, and hybrid structures were studied. The model glycoproteins included ribonuclease B, fetuin, α1-acid glycoprotein, immunoglobulin, and thyroglobulin. All N-glycans associated with these model glycoproteins were detected using the online PNGase F reactor setup.

Original languageEnglish
Pages (from-to)8790-8796
Number of pages7
JournalAnalytical Chemistry
Volume84
Issue number20
DOIs
StatePublished - Oct 16 2012

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