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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

Procedures of LC-MS/MS and RF-MS/MSBefore application to LC-MS/MS or RF-MS/MS, all the procedures were performed in the same manner.
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Figure 3: Procedures of LC-MS/MS and RF-MS/MSBefore application to LC-MS/MS or RF-MS/MS, all the procedures were performed in the same manner.

Mentions: The instrument used for high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) was a 1260 infinity LC/6460 Triple Quad (Agilent Technologies, Palo Alto, CA, USA). Samples were removed from the -20°C and were allowed to thaw at an ambient temperature. Sample tubes were quickly spun by using a VWR Galaxy Ministar centrifuge to collect all samples to the bottom of the tube. Twenty-five μL of each sample was transferred to a 96 well polypropylene plate, and diluted with 25 μL of 50 mMTris buffer (pH 7.5). The plate was centrifuged at 3,500 g for 5 min prior to analysis with the 1260 infinity LC /6460 Triple Quad (Figure 3).


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)

Procedures of LC-MS/MS and RF-MS/MSBefore application to LC-MS/MS or RF-MS/MS, all the procedures were performed in the same manner.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Procedures of LC-MS/MS and RF-MS/MSBefore application to LC-MS/MS or RF-MS/MS, all the procedures were performed in the same manner.
Mentions: The instrument used for high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) was a 1260 infinity LC/6460 Triple Quad (Agilent Technologies, Palo Alto, CA, USA). Samples were removed from the -20°C and were allowed to thaw at an ambient temperature. Sample tubes were quickly spun by using a VWR Galaxy Ministar centrifuge to collect all samples to the bottom of the tube. Twenty-five μL of each sample was transferred to a 96 well polypropylene plate, and diluted with 25 μL of 50 mMTris buffer (pH 7.5). The plate was centrifuged at 3,500 g for 5 min prior to analysis with the 1260 infinity LC /6460 Triple Quad (Figure 3).

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