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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

Comparison of disaccharide elution between filtersThe peak corresponding to Di-4S (DS) in the chromatogram after use of the Ultra filtration Omega 10K filter was much larger than seen for the other filters. The amount of KS1 (mono-sulfated KS) and KS2 (di-sulfated KS) (right panel) was also higher from this filter than the neonatal screening filter (note scale of y-axis). Elution of HS was similar for all 3 filters (data not shown).*AcroPrep™ Advance 96-Well Filter Plates.
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Figure 2: Comparison of disaccharide elution between filtersThe peak corresponding to Di-4S (DS) in the chromatogram after use of the Ultra filtration Omega 10K filter was much larger than seen for the other filters. The amount of KS1 (mono-sulfated KS) and KS2 (di-sulfated KS) (right panel) was also higher from this filter than the neonatal screening filter (note scale of y-axis). Elution of HS was similar for all 3 filters (data not shown).*AcroPrep™ Advance 96-Well Filter Plates.

Mentions: Purified polymer CS (shark cartilage), DS (pig skin), HS (bovine kidney), and KS (bovine cornea) were digested by a mixture of chondroitinase C (Sigma-Aldrich Co, St. Louis MO), chondroitinase B, heparitinase, and keratanase II (all other reagents except chondroitinase C provided by Seikagaku Co). Samples to be measured were centrifuged to remove insoluble material. Ten μL of the supernatant was mixed with 90 μL of 50 mM Tris-HCl Buffer (pH 7.0), and then added to a filter well. This was centrifuged at 2,500 g for 15 min to deplete low molecular compounds that could interfere with the assay. The filters were transferred to fresh plates and 20 μL of internal standard (500 ng/mL chondrosine), 20 μL of 50 mM Tris-HCl buffer and 10 μL of chondroitinase C, chondroitinase B, heparitinase, and keratanase II, respectively, (each 2 mU/10 μL of 50 mM Tris-HCl buffer) were added onto each filter. Samples were incubated with shaking at 37°C for 15 hr to digest the GAGs. Filter plates were then centrifuged at 2,500 g for 15 min. Twenty μL of ddH2O was added to each flow-through sample, and mixed by vortexing for 10 sec. Samples were stored at -20°C until injection to LC-MS/MS or RF-MS/MS. In a preliminary study we tested three different filter systems for efficiency of extraction and recovery: AcroPrep™ Advance 96-Well Filter Plates, with Ultra filtration Omega 10K(PALL Co., NY, USA); Neonatal Screening, AcroPrept™ Advance 96-Well Filter Plates (PALL Co.); and Amicon Ultra 10K device (Milipore Co.) and monitored recovery of Di4S and KS (Figure 2). The optimized Di-4S assay also detects Di-6S because these disaccharides have the same molecular mass and yield similar fragments. The KS assay detects both mono and disulfated KS because the fragmentor can remove a sulfate from the disulfated form before it enters the mass spectrometer. The filter with the ultrafiltration Omega 10K filters gave the highest yield of disaccharides, especially for Di-4S.


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)

Comparison of disaccharide elution between filtersThe peak corresponding to Di-4S (DS) in the chromatogram after use of the Ultra filtration Omega 10K filter was much larger than seen for the other filters. The amount of KS1 (mono-sulfated KS) and KS2 (di-sulfated KS) (right panel) was also higher from this filter than the neonatal screening filter (note scale of y-axis). Elution of HS was similar for all 3 filters (data not shown).*AcroPrep™ Advance 96-Well Filter Plates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Comparison of disaccharide elution between filtersThe peak corresponding to Di-4S (DS) in the chromatogram after use of the Ultra filtration Omega 10K filter was much larger than seen for the other filters. The amount of KS1 (mono-sulfated KS) and KS2 (di-sulfated KS) (right panel) was also higher from this filter than the neonatal screening filter (note scale of y-axis). Elution of HS was similar for all 3 filters (data not shown).*AcroPrep™ Advance 96-Well Filter Plates.
Mentions: Purified polymer CS (shark cartilage), DS (pig skin), HS (bovine kidney), and KS (bovine cornea) were digested by a mixture of chondroitinase C (Sigma-Aldrich Co, St. Louis MO), chondroitinase B, heparitinase, and keratanase II (all other reagents except chondroitinase C provided by Seikagaku Co). Samples to be measured were centrifuged to remove insoluble material. Ten μL of the supernatant was mixed with 90 μL of 50 mM Tris-HCl Buffer (pH 7.0), and then added to a filter well. This was centrifuged at 2,500 g for 15 min to deplete low molecular compounds that could interfere with the assay. The filters were transferred to fresh plates and 20 μL of internal standard (500 ng/mL chondrosine), 20 μL of 50 mM Tris-HCl buffer and 10 μL of chondroitinase C, chondroitinase B, heparitinase, and keratanase II, respectively, (each 2 mU/10 μL of 50 mM Tris-HCl buffer) were added onto each filter. Samples were incubated with shaking at 37°C for 15 hr to digest the GAGs. Filter plates were then centrifuged at 2,500 g for 15 min. Twenty μL of ddH2O was added to each flow-through sample, and mixed by vortexing for 10 sec. Samples were stored at -20°C until injection to LC-MS/MS or RF-MS/MS. In a preliminary study we tested three different filter systems for efficiency of extraction and recovery: AcroPrep™ Advance 96-Well Filter Plates, with Ultra filtration Omega 10K(PALL Co., NY, USA); Neonatal Screening, AcroPrept™ Advance 96-Well Filter Plates (PALL Co.); and Amicon Ultra 10K device (Milipore Co.) and monitored recovery of Di4S and KS (Figure 2). The optimized Di-4S assay also detects Di-6S because these disaccharides have the same molecular mass and yield similar fragments. The KS assay detects both mono and disulfated KS because the fragmentor can remove a sulfate from the disulfated form before it enters the mass spectrometer. The filter with the ultrafiltration Omega 10K filters gave the highest yield of disaccharides, especially for Di-4S.

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