Mannose 6-phosphate quantitation in glycoproteins using high-pH anion-exchange chromatography with pulsed amperometric detection.
An assay has been developed to quantitate the amount of mannose 6-phosphate in glycoproteins using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The method was tested on a recombinant lysosomal enzyme, human alpha-galactosidase A, that contains mannose 6-phosphate. The assay includes two steps: hydrolysis of the glycoprotein in 6.75 M trifluoroacetic acid to release mannose 6-phosphate and quantitation of the released mannose 6-phosphate using HPAEC with PAD. There is a linear relationship between the amount of mannose 6-phosphate measured and the amount of alpha-galactosidase hydrolyzed. The assay is also sensitive for as little as 2.5 microg alpha-galactosidase, which contains 117 pmol mannose 6-phosphate. Further, the assay has been shown to have good day-to-day and operator-to-operator consistency. In order to evaluate the assay for glycoprotein in crude extract, the glycoprotein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The amount of mannose 6-phosphate in the electroblots following hydrolysis was determined using HPAEC-PAD. The assay was also linear when measuring mannose 6-phosphate on electroblots. Therefore, this assay has been shown to be specific, sensitive, and reproducible.
Zhou
Qun
Q
Structural Protein Chemistry, Genzyme Corporation, Framingham, Massachusetts 01701, USA.
Kyazike
Josephine
J
Edmunds
Tim
T
Higgins
Elizabeth
E
D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D006023:Glycoproteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008360:Mannosephosphates / Q000032:analysis*; D011145:Polyvinyls; D005465:Fluorocarbon Polymers
D006023:Glycoproteins; D008360:Mannosephosphates; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C027693:mannose-6-phosphate