TY - JOUR
T1 - Fast and efficient online release of N-glycans from glycoproteins facilitating liquid chromatography-tandem mass spectrometry glycomic profiling
AU - Jmeian, Yazen
AU - Hammad, Loubna A.
AU - Mechref, Yehia
N1 - Copyright:
Copyright 2013 Elsevier B.V., All rights reserved.
PY - 2012/10/16
Y1 - 2012/10/16
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84869398333&partnerID=8YFLogxK
U2 - 10.1021/ac301855v
DO - 10.1021/ac301855v
M3 - Article
C2 - 22978794
AN - SCOPUS:84869398333
VL - 84
SP - 8790
EP - 8796
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 20
ER -