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

Abstract

We examined the analysis of nucleotides and nucleotide sugars by chromatography on porous graphitic carbon with mass spectrometric detection, a method that evades contamination of the MS instrument with ion pairing reagent. At first, adenosine triphosphate (ATP) and other triphosphate nucleotides exhibited very poor chromatographic behavior on new columns and could hardly be eluted from columns previously cleaned with trifluoroacetic acid. Satisfactory performance of both new and older columns could, however, be achieved by treatment with reducing agent and, unexpectedly, hydrochloric acid. Over 40 nucleotides could be detected in cell extracts including many isobaric compounds such as ATP, deoxyguanosine diphosphate (dGTP), and phospho-adenosine-5′-phosphosulfate or 3′,5′-cyclic adenosine 5'-monophosphate (AMP) and its much more abundant isomer 2′,3′-cylic AMP. A fast sample preparation procedure based on solid-phase extraction on carbon allowed detection of very short-lived analytes such as cytidine 5'-monophosphate (CMP)-2-keto-deoxy-octulosonic acid. In animal cells and plant tissues, about 35 nucleotide sugars were detected, among them rarely considered metabolites such as uridine 5'-diphosphate (UDP)-l-arabinopyranose, UDP-l-arabinofuranose, guanosine 5'-diphosphate (GDP)-l-galactofuranose, UDP-l-rhamnose, and adenosine diphosphate (ADP)-sugars. Surprisingly, UDP-arabinopyranose was also found in Chinese hamster ovary (CHO) cells. Due to the unique structural selectivity of graphitic carbon, the method described herein distinguishes more nucleotides and nucleotide sugars than previously reported approaches.

Renal 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase Is an Important Determinant of AKI Severity after Ischemia-Reperfusion

“…The production of 29,39-cAMP as well as other nucleoside 29,39-cyclic monophosphates by living tissues has been corroborated by multiple independent laboratories. [26][27][28][29][30][31] Our hypothesis that injury increases 29,39-cAMP production also has received confirmation. For example, brain trauma in mice increases brain interstitial levels of 29,39-cAMP, and 29,39-cAMP is elevated in cerebrospinal fluid of humans in the early hours after traumatic brain injury.…”

The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis

“…Consequently, to characterize the URGT family of Arabidopsis fully, we enzymatically synthesized nucleotide sugars, such as UDP-Rha and GDP-Gal, which are not currently commercially available in either the unlabeled or radiolabeled form. To overcome the limitations of using radiolabeled substrates, we adapted and optimized a recently developed LC-MS method, which facilitates the separation and quantification of all major nucleotide sugars (34). In conjunction with this MSbased assay technique, we reconstituted each member of the NST-KT clade into preloaded liposomes to determine its substrate specificity using a mix of 13 nucleotide sugars.…”

“…Analysis of Nucleotide Sugars by MS. Nucleotide sugar separations were undertaken using porous graphitic carbon as the stationary phase using an Agilent 1100 Series HPLC instrument coupled to a mass spectrometer (34) (SI Materials and Methods). Detection was undertaken using a 4000 QTRAP LC-MS/MS system (AB Sciex) equipped with a TurboIonSpray ion source (AB Sciex) (SI Materials and Methods).…”

Activated Sugar Precursors: Biosynthetic Pathways and Biological Roles of an Important Class of Intermediate Metabolites in Bacteria

“…Capillary electrophoresis (CE) has also been used to resolve closely related sugar nucleotides, together with NMR spectroscopy to identify their chemical structures (Lehmann et al, 2000;King et al, 2009). Recently, porous graphitic carbon (PGC) liquid chromatographyelectrospray ionization-mass spectrometry (LC-ESI-MS) was successfully applied to sugar nucleotide separation and analysis (Pabst et al, 2010).…”