Packages
Work Package 8
Analysis of oligosaccharides in urine and serum of patients and mice
- Involves Partners ZYMX, CAU, CUH, CMMC, UMG, HCL, DTI and IPCZD
Objectives:
The aim of this WP is to determine and optimize the oligosascharide structure analysis of urine and serum of ERT treated alpha-Mannosidosis patients and mice. In addition, this WP will perform detailed glycostructure analysis of the highly purified recombinant enzyme, which is used for the clinical trials in man and in chronic treatment studies in mice.
Description of the Work:
Within HUE-MAN optimal conditions and protocol for the qualitative (MALDI-TOF) and quantitative (HPLC) analyses of oligosaccharides from tissues, urine and plasma were developed. The developed methods allow a precise determination of both i) the nature and ii) the quantity of each remnant oligomannoside isomer family (e.g. Man2 to Man9) in mouse tissues as well as in urine and serum samples of alpha-Mannosidosis patients. The oligosaccharide content in urine and serum of patients before and after ERT is an important biomarker for the clinical trials, to assess the efficacy of ERT in alpha-Mannosidosis patients. Additionally, it will also help to study the carbohydrate content in tissues of ERT treated immune-tolerant alpha-Mannosidosis mice.
Qualitative Analysis:
For qualitative sugar analyses with highest sensitivity, oligosaccharides from mouse tissues and patients urine and serum samples are permethylated, purified on a Sep Pack C18 and analysed by MALDI-TOF-MS. MALDI currently is the ionization method of choice for mass fingerprinting. Structural determination of the oligos is performed using q-TOF or Orbitrap MS analysis. Using these approaches, the overall carbohydrate content of the sample and relative abundance of specific moieties such as high mannose type oligosaccharides can be determined.
Quantification Analysis:
To quantify high-mannose type oligosaccharides in patients and mice samples, an HPLC analysis will be performed with an internal standard, that is added to the sample prior oligosaccharide purification. Oligosaccharides are purified on a Sep Pack C18 and labeled with a fluorescent tag so that the labeled oligosaccharides can then be separated using normal phase chromatography with fluorescence detection. Since carbohydrates do not contain chromophores suitable for any easy detection by nature they have to be labelled with fluorescent tags such as 2-aminobenzamide. The fluorescence of this tag was then used to quantify these oligosaccharides.