Direct Determination of Mannose-6-Phosphate Content of Glycoproteins

June 1, 2009

The Application Notebook

The Application Notebook, The Application Notebook-06-01-2009, Volume 0, Issue 0

D-Mannose-6-phosphate (M-6-P) is a terminal monosaccharide of some asparagine-linked (N-linked) oligosaccharides and is also part of an important intermediate in N-linked oligosaccharide biosynthesis. Some lysosomal glycoproteins require M-6-P terminated oligosaccharides for proper targeting and function. Lack of M-6-P or genetic defects in its synthesis or subsequent processing can result in a variety of diseases.

Terri T. Christison, Brian M. De Borba, and Jeffrey S. Rohrer, Dionex Corporation

D-Mannose-6-phosphate (M-6-P) is a terminal monosaccharide of some asparagine-linked (N-linked) oligosaccharides and is also part of an important intermediate in N-linked oligosaccharide biosynthesis. Some lysosomal glycoproteins require M-6-P terminated oligosaccharides for proper targeting and function. Lack of M-6-P or genetic defects in its synthesis or subsequent processing can result in a variety of diseases.

High performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) is a well-established technique for determining a large variety of carbohydrates, including M-6-P (1). The HPAE-PAD method described here accurately determines M-6-P added to an acid-hydrolyzed protein.

Experimental

A Dionex® ICS-3000 Ion Chromatography system with a CarboPac® PA200 (3 × 250 mm) analytical column was used. M-6-P was separated from the components of acid hydrolyzed bovine serum albumin (BSA) with 100 mM sodium hydroxide and 100 mM sodium acetate at 30 °C, a flow rate of 0.5 mL/min, and detected by PAD as described in reference (2).

Figure 1

Results

HPAE-PAD is a selective, sensitive, and accurate method for determining the M-6-P content of a glycoprotein without derivatization (1). Here, we demonstrate an updated HPAE-PAD method for M-6-P determinations using a newer column and improved electrochemical conditions. Because a M-6-P-containing glycoprotein was not commercially available, we hydrolyzed BSA in 6.75 M trifluoroacetic acid (TFA) for 1.5 h at 100 °C (1), and added M-6-P after hydrolysis. Figure 1 shows that M-6-P is well resolved from the components of the hydrolyzed BSA and accurately determined (101 and 104% recoveries). To confirm the M-6-P peak, we treated the two samples with alkaline phosphatase, re-assayed the samples, and assayed the samples for mannose with another HPAE-PAD method (2). The M-6-P peak in each sample disappeared (not shown) and mannose was recovered (96.5 and 96.8%) (Figure 2). Therefore, HPAE-PAD can accurately determine M-6-P in an acid hydrolyzed glycoprotein sample and confirm M-6-P presence by both substrate disappearance and product appearance.

Figure 2

References

(1) Zhou, Q., Kyazike, J., Edmunds, T., Higgins, E. Anal. Biochem., 2002, 306, 163–170.

(2) Dionex Corporation. Application Note 202, LPN 2076. Sunnyvale, CA, USA, 2008.

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