title: High information throughput analysis of nucleotides and their isotopically enriched isotopologues by direct-infusion FTICR-MS.
abstract: Fourier transform-ion cyclotron resonance-mass spectrometry (FTICR-MS) is capable of acquiring unmatched quality of isotopologue data for stable isotope resolved metabolomics (SIRM). This capability drives the need for a continuous ion introduction for obtaining optimal isotope ratios. Here we report the simultaneous analysis of mono and dinucleotides from crude polar extracts by FTICR-MS by adapting an ion-pairing sample preparation method for LC-MS analysis. This involves a rapid cleanup of extracted nucleotides on pipet tips containing a C(18) stationary phase, which enabled global analysis of nucleotides and their (13)C isotopologues at nanomolar concentrations by direct infusion nanoelectrospray FTICR-MS with 5 minutes of data acquisition. The resolution and mass accuracy enabled computer-assisted unambiguous assignment of most nucleotide species, including all phosphorylated forms of the adenine, guanine, uracil and cytosine nucleotides, NAD(+), NADH, NADP(+), NADPH, cyclic nucleotides, several UDP-hexoses, and all their (13)C isotopologues. The method was applied to a SIRM study on human lung adenocarcinoma A549 cells grown in [U-(13)C] glucose with or without the anti-cancer agent methylseleninic acid. At m/z resolving power of 400,000, (13)C-isotopologues of nucleotides were fully resolved from all other elemental isotopologues, thus allowing their (13)C fractional enrichment to be accurately determined. The method achieves both high sample and high information throughput analysis of nucleotides for metabolic pathway reconstruction in SIRM investigations.
authors.affiliation: Department of Chemistry, University of Louisville, 2210 S. Brook St, Rm 348 John W. Shumaker Research Building, Louisville, KY 40292, USA.
authors.forename: Pawel
authors.initials: P
authors.lastname: Lorkiewicz
authors.forename: Richard M
authors.initials: RM
authors.lastname: Higashi
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Lane
authors.forename: Teresa W-M
authors.initials: TW
authors.lastname: Fan