Selective detection of sugar phosphates by capillary electrophoresis/mass spectrometry and its application to an engineered E. coli host.
A highly selective method employing capillary electrophoresis and electrospray mass spectrometry (CE-ESMS) with precursor ion scanning for fragment ions characteristic of phosphate-linked sugars was developed for the determination of "unnatural" sugar phosphates generated in vivo, as part of a natural product glycorandomization study. Cell lysates from an engineered E. coli host were probed for "natural" and "unnatural" sugar phosphates resulting from in vivo galactokinase (GalK) bioconversions, and tandem mass spectrometry experiments were performed to confirm the identities of the sugar phosphates. Among the 22 cell lysates that were studied, 13 were found to contain the expected natural and "unnatural" sugar phosphates. This was in agreement with the GalK in vitro conversion yields, in which an in vitro yield of <or=15 % coincided with a lack of observable in vivo bioconversion. In addition, the CE-ESMS and precursor ion scanning method was capable of separating sugar phosphate regioisomers such as hexose-6-phosphate and hexose-1-phosphate.
Hui
Joseph P M
JP
MS Metabolomics Group, NRC-Institute for Marine Biosciences, 1411 Oxford Street, Halifax, NS B3H 3Z1, Canada.
Yang
Jie
J
Thorson
Jon S
JS
Soo
Evelyn C
EC
D002241:Carbohydrates / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005686:Galactokinase / Q000378:metabolism; D005818:Genetic Engineering; D013058:Mass Spectrometry / Q000379:methods*; D008956:Models, Chemical; D010710:Phosphates / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods; D013997:Time Factors
D002241:Carbohydrates; D010710:Phosphates; D005686:Galactokinase