Analysis: Activated Nucleotide Sugars by LC-MS/MS

Tina Wigger, Scientific Expert

MilliporeSigma

Article from Analytix Reporter - Issue 14

Introduction

Nucleotide sugars, or nucleotide activated sugars, are highly energetic forms of monosaccharides that act as key metabolites in glycosylation reactions, during which the glycosyl group from the activated sugars is transferred to an acceptor molecule, e.g. a protein.

In mammals, the most common nucleotide for sugar activation is uridine diphosphate (UDP), which is found in combination with Glucose (Glc), Galactose (Gal), N-Acetylglucosamine (GlcNAc), N-Acetylgalactosamine (GalNAc), Glucuronic acid (GlcA) and Xylose (Xyl). Furthermore, Guanosine diphosphate (GDP) linked to Mannose (Man) and Fucose (Fuc) as well as Cytidine monophosphate (CMP) linked to sialic acid (Neu5Ac) are used for glycan assembly. Plants and bacteria utilize an even larger variety of nucleotides and sugars.

Understanding the nucleotide sugar metabolism is of interest in different scientific areas, e.g. for the production of glycosylated therapeutic proteins in cell culture, where a sufficient glycosylation needs to be ensured in order to obtain a product of high quality and efficacy.¹ For this purpose, a sensitive, quantification method is needed that is capable of simultaneously analyzing a set of polar analytes with similar structures and physicochemical properties. In this application note, an LC method using a Supel™ Carbon LC column in combination with selective MS/MS detection is described.

Experimental Conditions

The used conditions for the analysis of 11 nucleotide activated sugars in a standard solution with a concentration of 80 µM each are described in Table 1 & 2.

Table 1. HPLC Conditions for analysis of 11 nucleotide activated sugars

Column	Supel™ Carbon LC, 50 x 3 mm I.D., 2.7 µm (59991-U)
Mobile phase	[A] Water + 0.5 % diethylamine, pH 10 (adjusted with acetic acid); [B] acetonitrile
Gradient	Time (minutes)	A%	B%
	0	100	0
	2.0	100	0
	12.5	90	10
	21.0	25	75
	26.0	25	75
	26.5	100	0
	33.0	100	0
Flow rate	0.4 mL/min
Column temp.	60 °C
Detector	MS/MS, negative mode (Instrument: Agilent 6495 Triple Quadrupole); for used transitions see Table 2
Injection	10 µL
Needle Wash Solution	Water + 5% isopropanol
Sample(s)	10 mM individual stock solutions of the activated sugar standards were prepared in water and mixed to yield a concentration of 80 µM each.

Table 2. MS/MS Transitions nucleotide activated sugars

Analyte	Q1 mass (m/z)	Q3 mass (m/z)
ADP-Glucose	588.1	346.0
ADP-Ribose	558.1	346.1
CMP-Neu5Ac	613.2	322.0
GDP-Fucose	588.1	442.1
GDP-Mannose	604.0	424.0
UDP-Galactose	565.0	323.0
UDP-GalNAc/UDP-GcNAc	606.1	385.1
UDP-Glucose	565.0	323.1
UDP-Glucuronic acid	579.0	403.1
UDP-Xylose	535.0	323.1
TDP-Glucose	563.1	321.1

Results

The 5 cm x 3 mm I.D. Supel™ Carbon LC column provided, under reversed phase conditions, good retention of the 11 activated nucleotide sugar compounds in the applied standard solution (Figure 1 & Table 3).

Figure 1. LC-MS/MS Analysis of nucleotide activated sugars on a Supel™ Carbon LC column.

Table 3. Peak identification and retention times of nucleotide activated sugars on the Supel™ Carbon LC column

Peak no.	Compound	Retention time (min)
1	CMP-Neu5Ac	12.75
2	UDP-Glucose	12.89
3	UDP-Glucuronic acid	12.91
4	UDP-Galactose	13.41
5	UDP-Xylose	13.49
6	UDP-GalNAc	13.62
7	GDP-Mannose	14.55
8	TDP-Glucose	14.65
9	GDP-Fucose	14.81
10	ADP-Ribose	15.05
11	ADP-Glucose	15.11

Conclusion

This application demonstrated the use and suitability of the Supel^™ Carbon LC column for the analysis of 11 nucleotide activated sugars. The group of highly polar compounds with similar structures and physicochemical properties was sufficiently retained and separated on the carbon LC column to be selectively detected with the described MS/MS method. Its ability to retain polar compounds makes the Supel^™ Carbon LC column a viable choice for the analysis and quantitation of nucleotide activated sugars under reversed phase (RP) conditions.

See more information about the Supel™ Carbon LC column at SigmaAldrich.com/carbonlc

References

1. Glycans in Biotechnology and the Pharmaceutical Industry. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 51. [Internet]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1908/