title: Analysis of nucleotides by pressure-assisted capillary electrophoresis-mass spectrometry using silanol mask technique.
abstract: A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis-electrospray ionization mass spectrometry (PACE-MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions, 14 phosphorylated species such as nucleotides, nicotinamide-adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations (n=6) of the method were better than 0.9% for migration times and between 1.7% and 8.1% for peak areas. The detection limits for these species were between 0.5 and 1.7 micromol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme.
authors.affiliation: Institute for Advanced Biosciences, Keio University, Kakuganji, Tsuruoka, Yamagata 997-0052, Japan. soga@sfc.keio.ac.jp
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Soga
authors.forename: Takamasa
authors.initials: T
authors.lastname: Ishikawa
authors.forename: Saori
authors.initials: S
authors.lastname: Igarashi
authors.forename: Kaori
authors.initials: K
authors.lastname: Sugawara
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kakazu
authors.forename: Masaru
authors.initials: M
authors.lastname: Tomita
mesh_terms: D003065:Coenzyme A / Q000032:analysis; D004573:Electrolytes; D053841:Electroosmosis; D019075:Electrophoresis, Capillary / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry; D009711:Nucleotides / Q000032:analysis*; D010710:Phosphates / Q000737:chemistry; D011312:Pressure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012821:Silanes / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D018511:Systems Integration
chemical_list: D004573:Electrolytes; D029968:Escherichia coli Proteins; D009711:Nucleotides; D010710:Phosphates; D012821:Silanes; C082343:silanol; D003065:Coenzyme A