Nucleotide and Nucleotide Sugar Analysis by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry on Surface-Conditioned Porous Graphitic Carbon

摘要

We examined the analysis of nucleotides and nucleotidensugars by chromatography on porous graphitic carbonnwith mass spectrometric detection, a method that evadesncontamination of the MS instrument with ion pairingnreagent. At first, adenosine triphosphate (ATP) and otherntriphosphate nucleotides exhibited very poor chromatographicnbehavior on new columns and could hardly beneluted from columns previously cleaned with trifluoroaceticnacid. Satisfactory performance of both new andnolder columns could, however, be achieved by treatmentnwith reducing agent and, unexpectedly, hydrochloric acid.nOver 40 nucleotides could be detected in cell extractsnincluding many isobaric compounds such as ATP, deoxyguanosinendiphosphate (dGTP), and phospho-adenosine-n5′-phosphosulfate or 3′,5′-cyclic adenosine 5′-monophosphaten(AMP) and its much more abundant isomer 2′,3′-ncylic AMP. A fast sample preparation procedure basednon solid-phase extraction on carbon allowed detection ofnvery short-lived analytes such as cytidine 5′-monophosphaten(CMP)-2-keto-deoxy-octulosonic acid. In animalncells and plant tissues, about 35 nucleotide sugars werendetected, among them rarely considered metabolites suchnas uridine 5′-diphosphate (UDP)-L-arabinopyranose, UDPL-narabinofuranose, guanosine 5′-diphosphate (GDP)-Lgalactofuranose,nUDP-L-rhamnose, and adenosine diphosphaten(ADP)-sugars. Surprisingly, UDP-arabinopyranosenwas also found in Chinese hamster ovary (CHO) cells. Duento the unique structural selectivity of graphitic carbon, thenmethod described herein distinguishes more nucleotidesnandnucleotidesugarsthanpreviouslyreportedapproaches.