Nucleotide and nucleotide sugar analysis by liquid chromatography-electrospray ionization-mass spectrometry on surface-conditioned porous graphitic carbon.
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'-cyclic 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.
Department of Chemistry, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
Martin
M
Pabst
Josephine
J
Grass
Richard
R
Fischl
Renaud
R
Léonard
Chunsheng
C
Jin
Georg
G
Hinterkörner
Nicole
N
Borth
Friedrich
F
Altmann
D000250:Adenosine Phosphosulfate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D002244:Carbon / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D006153:Guanosine Diphosphate / Q000737:chemistry; D007536:Isomerism; D009711:Nucleotides / Q000032:analysis*; D016062:Porosity; D019163:Reducing Agents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014539:Uridine Diphosphate Sugars / Q000737:chemistry
D009711:Nucleotides; D019163:Reducing Agents; D014539:Uridine Diphosphate Sugars; D006153:Guanosine Diphosphate; D000250:Adenosine Phosphosulfate; D002244:Carbon; D000255:Adenosine Triphosphate