Ion-pairing liquid chromatography–tandem mass spectrometry-based quantification of uridine diphosphate-linked intermediates in the Staphylococcus aureus cell wall biosynthesis pathway

2014 ◽  
Vol 465 ◽  
pp. 12-19 ◽  
Author(s):  
Harika Vemula ◽  
Sudheer Bobba ◽  
Sandeep Putty ◽  
Joanna E. Barbara ◽  
William G. Gutheil
Keyword(s):  
Mass Spectrometry ◽  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Liquid Chromatography ◽  
Cell Wall Biosynthesis ◽  
Tandem Mass ◽  
Uridine Diphosphate ◽  
Biosynthesis Pathway ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

scholarly journals Liquid Chromatography–Tandem Mass Spectrometry Enzyme Assay for UDP-Galactose 4′-Epimerase: Use of Fragment Intensity Ratio in Differentiation of Structural Isomers

2014 ◽  
Vol 60 (5) ◽  
pp. 783-790 ◽  
Author(s):  
Yijun Li ◽  
Xiaoping Huang ◽  
Lauren Harmonay ◽  
Ying Liu ◽  
Mark D Kellogg ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Tandem Mass ◽  
Uridine Diphosphate ◽  
Structural Isomers ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Diphosphate Glucose ◽  
Clinically Significant

Abstract BACKGROUND Distinction between asymptomatic and potentially clinically significant forms of galactosemia due to UDP-galactose 4′-epimerase (GALE) deficiency requires enzyme measurement in erythrocytes and other cells. We sought to develop a GALE assay using a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based method. METHODS The reversible GALE assay was conducted with UDPGal as a substrate. The coeluting reaction product, uridine diphosphate glucose (UDPGlc), and its isomeric substrate, uridine diphosphate galactose (UDPGal), were detected by MS/MS at mass transitions 565 > 280, 565 > 241 and 565 > 403. The UDPGal was enriched in mass transition 565 > 403 compared with UDPGlc, whereas the UDPGlc was enriched in the mass transition 565 > 241 compared with UDPGal. The percentage of UDPGal in the reaction mixture was calculated by use of the ratio of ion intensities of the 2 daughter ions and a fourth-order polynomial calibrator curve. RESULTS The method yielded a mean (SD) GALE activity of 9.8 (2.2) μmol · g−1 hemoglobin · h−1 in erythrocyte extracts from 27 controls. The apparent Km of the substrate, UDPGal, was 0.05 mmol/L. The GALE activity ranged from 433 to 993 μmol · g−1 protein · h−1 in control lymphoblast extracts. In a blinded test of 22 subjects suspected of GALE deficiency, we identified 6 individuals whose residual activities were below the range of controls, compatible with intermediate GALE deficiency. CONCLUSIONS This assay can be used to distinguish the different forms of GALE deficiency. From an analytical standpoint, differentiating isomers on the basis of fragment intensity ratios should also prove useful for analogous enzymatic studies involving substrates and products that are structural isomers.

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