Direct-infusion FT-ICR MS method for the analysis of nucleotides and their labeled isotopologues in crude polar extracts

摘要

Direct-infusion Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has been used for simultaneous analysis of amino acids, lipids and other metabolites in metabolomic studies. To date direct-infusion analysis of nucleotides and nucleotide sugars has been difficult, partly because of high salt content and ion suppression effects of the sample matrix in crude polar extracts. The low S/N typically obtained for nucleotide metabolites makes it even more difficult to determine their isotopic labeling patterns in stable isotope-resolved metabolomic (SIRM) studies. To address these issues, we developed a convenient method based on reaction with ion-pairing reagent (IPR), followed by isolation and clean up of nucleotides using C18 stationary phase bed and simultaneous detection using direct-infusion FT-ICR MS. Nucleotide standards and lyophilized crude polar extracts of A549 and H1299 lung cancer cells, mouse, and human tissues were dissolved in 5 mM hexylamine aqueous solution (pH 6.3). The nucleotides in the extracts were first adsorbed onto the C18 stationary phase embedded in pipette tips (ZipTips~(~R) - Millipore) by consecutive aspirations. The nucleotide fraction was eluted by washing the tip with two aliquots of 70percent ion-pair solution and 30percent buffer consisting of 1 mM ammonium acetate in methanol (pH 8.5). The combined elution fractions were diluted in methanol (typically 3-fold) and analyzed directly with automated nanoelectrospray source (Triversa Nanomate~(~R), Advion) coupled to LTQ~(~R)-FT-ICR MS (Thermo). The efficacy of the method was first tested on a mixture of nucleotide standards dissolved in methanol. All components were detected simultaneously and the mass spectra were similar to those obtained for the same mixture without the use of the tip. Next, the method was evaluated with polar extracts of A549 and H1299 cancer cells. When MS analysis was done directly for extracts without the method, most of the nucleotides could not be detected. In addition, it was difficult to establish stable nanospray conditions. In contrast, when polar extracts were pre-processed with C18 tips, the common nucleotides and nucleotide sugars were detected with high sensitivity for both A549 and H1299 extracts. Salt effects and ion-suppression from sample matrix observed without IPR and C18 cleanup were greatly decreased. Moreover, stable nanospray conditions were established to enable 5 minute or longer acquisition times. The method was also applied to polar extracts of mouse and human lung tissues. Despite the higher salt content compared to cell extracts, common nucleotides were readily observed in the tissue extracts. The high S/N of nucleotide signals achieved using the present method allowed for determination of stable isotope labeling patterns by FT-ICR MS in experiments using tracers such as [U-~(13)C]-glucose. The mass spectra of labeled A549 and H1299 cell extracts clearly showed incorporation of ~(13)C from [U-~(13)C]-glucose into all detected nucleotides. Utilizing the high-resolving power of FT-ICR MS, it was practical to accurately quantify the complex isotopologue distributions of various nucleotides in these extracts. Positional atom incorporation was independently verified by NMR. Therefore the present method can be applied generally in SIRM studies for the determination of detailed stable isotope labeling patterns in nucleotides and nucleotide sugars, which can in turn be used for reconstruction of pathways and flux modeling involving these metabolites.