Determination of glycosaminoglycan levels by mass spectrometry
Detecting glycosaminoglycans (GAGs) and/or determining the level of one or more glycosaminoglycans can be useful, e.g., in identifying or monitoring various medical conditions, the status of patients having various medical conditions, and/or the response to treatment of individuals having various medical conditions. The present invention provides methods for detecting glycosaminoglycans and/or determining the level of glycosaminoglycans through the use of, e.g., mass spectrometry.
1. A method of determining glycosaminoglycan (GAG) level in a biological sample, the method comprising the steps of:
a) incubating a biological sample with one or more enzymes under conditions that permit digestion of glycosaminoglycan to generate a mixture of disaccharides;
b) chemically derivatizing the mixture of disaccharides generated in step a);
c) measuring an amount of each individual derivatized disaccharide;
d) determining the glycosaminoglycan (GAG) level in the biological sample based on the amount of each individual derivatized disaccharide measured at step c), wherein the step c) comprises
(i) separating individual derivatized disaccharides by chromatography; and
(ii) measuring each individual derivatized disaccharide by mass spectrometry, and wherein the amount of each individual derivatized disaccharide is measured as compared with an internal standard, and wherein each individual disaccharide is derivatized with 4-Butylaniline and the internal standard for each corresponding disaccharide is labeled with 13 C 6 -4-Butylaniline.
2. The method of claim 1 , wherein the glycosaminoglycan comprises heparan sulfate.
3. The method of claim 1 , wherein the one or more enzymes comprise one or more heparinases.
4. The method of claim 3 , wherein the one or more heparinases comprises heparinases I, II and/or III.
5. The method of claim 1 , wherein the one or more enzymes comprise one or more enzymes selected from the group consisting of chondroitinase AC, chondroitinase B, chondroitinase C, Chondroitinase ABC and keratanases.
6. The method of claim 1 , wherein the mixture of disaccharides comprises disaccharides I-S (ΔUA,2S-GlcNS,6S), II-S (ΔUA-GlcNS,6S), III-S (ΔUA,2SGlcNS), IV-S (ΔUA-GlcNS), II-A (ΔUA-GlcNAc,6S), and/or IV-A (ΔUA-GlcNAc).
7. The method of claim 6 , wherein the measuring comprises measuring the amount of each of derivatized I-S (ΔUA,2S-GlcNS,6S), derivatized II-S (ΔUA-GlcNS,6S), derivatized III-S (ΔUA,2S-GlcNS), derivatized IV-S (ΔUA-GlcNS), derivatized II-A (ΔUA-GlcNAc,6S), and derivatized IV-A (ΔUA-GlcNAc).
8. The method of claim 1 , wherein the mixture of disaccharides is derivatized with a hydrophobic moiety.
9. The method of claim 1 , wherein the chromatography is reverse-phase liquid chromatography.
10. The method of claim 1 , wherein the glycosaminoglycan (GAG) level in the biological sample is determined by summed disaccharide concentration value based on the amount of each individual disaccharide measured.
11. The method of claim 1 , wherein the biological sample is a cerebrospinal fluid (CSF) sample, a blood sample, a plasma sample, or a urine sample.