title: Nanoscale ion-pair reversed-phase HPLC-MS for sensitive metabolome analysis.
abstract: In this article, we introduce a method using nanoscale ion-pair reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC) hyphenated to nanoelectrospray ionization high-resolution mass spectrometry (nano-ESI-HRMS) to separate and identify metabolites in cell extracts. Separation of metabolites was performed on a 100 μm i.d. C18 column with tributylamine (TBA) as the ion-pairing reagent and methanol as the eluent. Basic pH (9.4) of the mobile phase was critical to achieve sufficient retention and sharp metabolite elution at a low concentration of TBA (1.7 mM). Limits of detection were determined for 54 standards with an LTQ-Orbitrap mass spectrometer to be in the upper attomole to low femtomole range for key metabolites such as nucleotides, phosphorylated sugars, organic acids, and coenzyme A thioesters in solvent as well as in a complex matrix. To further evaluate the method, metabolome analysis was performed injecting different amounts of biomass of the methylotroph model organism Methylobacterium extorquens AM1. A (12)C/(13)C labeling strategy was implemented to improve metabolite identification. Analysis of three biological replicates performed with 1.5 ng of cell dry weight biomass equivalents resulted in the identification of 20 ± 4 metabolites, and analysis of 150 ng allowed identifying 157 ± 5 metabolites from a large spectrum of metabolite classes.
authors.affiliation: Institute of Microbiology, ETH Zurich, Zurich, Switzerland.
authors.forename: Patrick
authors.initials: P
authors.lastname: Kiefer
authors.forename: Nathanaël
authors.initials: N
authors.lastname: Delmotte
authors.forename: Julia A
authors.initials: JA
authors.lastname: Vorholt
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000295:instrumentation / Q000379:methods*; D023281:Genomics / Q000379:methods*; D055442:Metabolome; D020601:Methylobacterium extorquens / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*