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Assay for Glycosaminoglycans by Tandem Mass Spectrometry and its Applications.

Tomatsu S, Shimada T, Mason RW, Kelly J, LaMarr WA, Yasuda E, Shibata Y, Futatsumori H, Montaño AM, Yamaguchi S, Suzuki Y, Orii T - J Anal Bioanal Tech (2014)

Bottom Line: The RF system consists of an integrated solid phase extraction robot that binds and de-salts samples from assay plates and directly injects them into a MS/MS detector, reducing sample processing time to ten seconds.However, the RF system does not have a chromatographic step, and therefore, cannot distinguish GAGs that have identical molecular weights.In this article, we compare the assay methods for GAGs and describe their potential applications.

View Article: PubMed Central - PubMed

Affiliation: Nemours/Alfred I. duPont Hospital for Children, Wilmington, DE, USA.

ABSTRACT
Glycosaminoglycans (GAGs) are distributed in the whole body and play a variety of important physiological roles associated with inflammation, growth, coagulation, fibrinolysis, lipolysis, and cell-matrix biology. Accumulation of undegraded GAGs in lysosomes gives rise to a distinct clinical syndrome, mucopolysaccharidoses. Measurement of each specific GAG in a variety of specimens is urgently required to understand GAG interaction with other molecules, physiological status of patients, and prognosis and pathogenesis of the disease. We established a highly sensitive and accurate tandem mass spectrometry (LC-MS/MS) method for measurements of disaccharides derived from four specific GAGs [dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS)]. Disaccharides were produced by specific enzyme digestion of each GAG, and quantified by negative ion mode of multiple reaction monitoring. Subclasses of HS and GAGs with identical molecular weights can be separated using a Hypercarbcolumn (2.0 mm×50 mm, 5 μm) with an aectonitrile gradient in ammonium acetate (pH 11.0). We also developed a GAG assay by RapidFire with tandem mass spectrometry (RF-MS/MS). The RF system consists of an integrated solid phase extraction robot that binds and de-salts samples from assay plates and directly injects them into a MS/MS detector, reducing sample processing time to ten seconds. RF-MS/MS consequently yields much faster throughput than conventional LC-MS/MS-based methods. However, the RF system does not have a chromatographic step, and therefore, cannot distinguish GAGs that have identical molecular weights. Both methods can be applied to analysis of dried blood spots, blood, and urine specimens. In this article, we compare the assay methods for GAGs and describe their potential applications.

No MeSH data available.


Related in: MedlinePlus

Chromatograms of disaccharidesChromatograms for disaccharides of KS 1 and 2 (digested bovine cornea), ΔDiHS-NS, ΔDiHS-0S, ΔDiHS-6S, ΔDi-4S (DS), ΔDi-6S (C6S), and chondrosine (IS). Polymer KS was separated with mono-sulfated KS [KS1: Galβ1-4GlcNAc(6S)] and di-sulfated KS [KS2: Gal(6S)β1- 4GlcNAc(6S)] after digestion by keratanase II. Equipment: 6460 Triple Quad MS/MS with 1260 infinity LC (Agilent Technologies). IS: Internal Standard.
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Figure 5: Chromatograms of disaccharidesChromatograms for disaccharides of KS 1 and 2 (digested bovine cornea), ΔDiHS-NS, ΔDiHS-0S, ΔDiHS-6S, ΔDi-4S (DS), ΔDi-6S (C6S), and chondrosine (IS). Polymer KS was separated with mono-sulfated KS [KS1: Galβ1-4GlcNAc(6S)] and di-sulfated KS [KS2: Gal(6S)β1- 4GlcNAc(6S)] after digestion by keratanase II. Equipment: 6460 Triple Quad MS/MS with 1260 infinity LC (Agilent Technologies). IS: Internal Standard.

Mentions: Disaccharides were separated on a Hypercarb (2.0 mm×50 mm, 5 μm; Thermo Electron Corp.) column and eluted with an acetonitrile gradient of 0% to 100% in 5 mM ammonium acetate. Signals for ΔDiHS-6S, ΔDi-4S (DS), and ΔDi-6S (C6S) were very low at pH values below 11 (Figure 4), probably due to poor ionization of the sugars. Using purified preparations ofΔDiHS-0S, ΔDiHS-NS, ΔDiHS-6S, ΔDi-4S, and ΔDi-6S, we optimized MRMs for each individual sugar and obtained single peaks corresponding to the pure compound (Figure 5). All disaccharides are eluted in less than 3 min (Figure 5). As discussed earlier, although ΔDiHS-6S, ΔDi-4S (DS), and ΔDi-6S (C6S) have the same molecular weights, they elute at different times from the column, so they can be quantified separately. Furthermore, the elution position of Di-4S varies dependent upon the sample preparation, and elutes at 0.9 min after extraction from some samples (e.g. Figure 4). All other disaccharides consistently eluted as seen for the standards. As discussed earlier, the detection method for mono-sulfated Galβ1-4GlcNAc(6S) (KS1) also detected some Gal(6S) β1-4GlcNAc(6S) (KS2), due to loss of a sulfate, but the two forms were clearly separated by chromatography.


Assay for Glycosaminoglycans by Tandem Mass Spectrometry and its Applications.

Tomatsu S, Shimada T, Mason RW, Kelly J, LaMarr WA, Yasuda E, Shibata Y, Futatsumori H, Montaño AM, Yamaguchi S, Suzuki Y, Orii T - J Anal Bioanal Tech (2014)

Chromatograms of disaccharidesChromatograms for disaccharides of KS 1 and 2 (digested bovine cornea), ΔDiHS-NS, ΔDiHS-0S, ΔDiHS-6S, ΔDi-4S (DS), ΔDi-6S (C6S), and chondrosine (IS). Polymer KS was separated with mono-sulfated KS [KS1: Galβ1-4GlcNAc(6S)] and di-sulfated KS [KS2: Gal(6S)β1- 4GlcNAc(6S)] after digestion by keratanase II. Equipment: 6460 Triple Quad MS/MS with 1260 infinity LC (Agilent Technologies). IS: Internal Standard.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109812&req=5

Figure 5: Chromatograms of disaccharidesChromatograms for disaccharides of KS 1 and 2 (digested bovine cornea), ΔDiHS-NS, ΔDiHS-0S, ΔDiHS-6S, ΔDi-4S (DS), ΔDi-6S (C6S), and chondrosine (IS). Polymer KS was separated with mono-sulfated KS [KS1: Galβ1-4GlcNAc(6S)] and di-sulfated KS [KS2: Gal(6S)β1- 4GlcNAc(6S)] after digestion by keratanase II. Equipment: 6460 Triple Quad MS/MS with 1260 infinity LC (Agilent Technologies). IS: Internal Standard.
Mentions: Disaccharides were separated on a Hypercarb (2.0 mm×50 mm, 5 μm; Thermo Electron Corp.) column and eluted with an acetonitrile gradient of 0% to 100% in 5 mM ammonium acetate. Signals for ΔDiHS-6S, ΔDi-4S (DS), and ΔDi-6S (C6S) were very low at pH values below 11 (Figure 4), probably due to poor ionization of the sugars. Using purified preparations ofΔDiHS-0S, ΔDiHS-NS, ΔDiHS-6S, ΔDi-4S, and ΔDi-6S, we optimized MRMs for each individual sugar and obtained single peaks corresponding to the pure compound (Figure 5). All disaccharides are eluted in less than 3 min (Figure 5). As discussed earlier, although ΔDiHS-6S, ΔDi-4S (DS), and ΔDi-6S (C6S) have the same molecular weights, they elute at different times from the column, so they can be quantified separately. Furthermore, the elution position of Di-4S varies dependent upon the sample preparation, and elutes at 0.9 min after extraction from some samples (e.g. Figure 4). All other disaccharides consistently eluted as seen for the standards. As discussed earlier, the detection method for mono-sulfated Galβ1-4GlcNAc(6S) (KS1) also detected some Gal(6S) β1-4GlcNAc(6S) (KS2), due to loss of a sulfate, but the two forms were clearly separated by chromatography.

Bottom Line: The RF system consists of an integrated solid phase extraction robot that binds and de-salts samples from assay plates and directly injects them into a MS/MS detector, reducing sample processing time to ten seconds.However, the RF system does not have a chromatographic step, and therefore, cannot distinguish GAGs that have identical molecular weights.In this article, we compare the assay methods for GAGs and describe their potential applications.

View Article: PubMed Central - PubMed

Affiliation: Nemours/Alfred I. duPont Hospital for Children, Wilmington, DE, USA.

ABSTRACT
Glycosaminoglycans (GAGs) are distributed in the whole body and play a variety of important physiological roles associated with inflammation, growth, coagulation, fibrinolysis, lipolysis, and cell-matrix biology. Accumulation of undegraded GAGs in lysosomes gives rise to a distinct clinical syndrome, mucopolysaccharidoses. Measurement of each specific GAG in a variety of specimens is urgently required to understand GAG interaction with other molecules, physiological status of patients, and prognosis and pathogenesis of the disease. We established a highly sensitive and accurate tandem mass spectrometry (LC-MS/MS) method for measurements of disaccharides derived from four specific GAGs [dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS)]. Disaccharides were produced by specific enzyme digestion of each GAG, and quantified by negative ion mode of multiple reaction monitoring. Subclasses of HS and GAGs with identical molecular weights can be separated using a Hypercarbcolumn (2.0 mm×50 mm, 5 μm) with an aectonitrile gradient in ammonium acetate (pH 11.0). We also developed a GAG assay by RapidFire with tandem mass spectrometry (RF-MS/MS). The RF system consists of an integrated solid phase extraction robot that binds and de-salts samples from assay plates and directly injects them into a MS/MS detector, reducing sample processing time to ten seconds. RF-MS/MS consequently yields much faster throughput than conventional LC-MS/MS-based methods. However, the RF system does not have a chromatographic step, and therefore, cannot distinguish GAGs that have identical molecular weights. Both methods can be applied to analysis of dried blood spots, blood, and urine specimens. In this article, we compare the assay methods for GAGs and describe their potential applications.

No MeSH data available.


Related in: MedlinePlus