High-Throughput, Accurate Mass Metabolome Profiling of Cellular Extracts by Flow Injection–Time-of-Flight Mass Spectrometry

Abstract

Direct injection of samples on high-resolving mass spectrometers is an effective way to maximize analytical throughput and yet allow analyte discrimination in complex samples by mass-to-charge ratio. We present a platform of flow injection electrospray-time-of-flight mass spectrometry to profile small molecules in >1400 biological extracts per day at native mass resolution. We comprehensively benchmark the performance with more than 5000 injections of chemically defined standards and Escherichia coli cellular extracts obtained from miniscale cultivations. For at least 90% of tested compounds, we attain a linear response over 3 decades of concentration, interday coefficient of variation of <20%, and a mass accuracy of <0.001 amu. In polar Escherichia coli fractions, we reproducibly detected >1500 distinct ions in each mode. The accurate mass and correlation analysis enabled one to assign with good confidence 400-800 ions to electrospray derivatives of metabolites listed in the genome-wide reconstruction of Escherichia coli metabolism. Hence, we attain a coverage of about one-quarter of the total number of compounds listed in the reconstruction. Finally, we present an exemplary screen with Escherichia coli deletion mutants to show the exquisite capacity of the platform to identify lesions in primary metabolism at high throughputs.

Ubiquinone accumulation improves osmotic-stress tolerance in Escherichia coli

“…1b). Using non-targeted flow injection time-of-flight MS 17 , we detected 8,961 ions of which 535 could be annotated based on accurate mass, corresponding to up to 1,071 unique metabolites. For further analyses we considered only the 535 annotated ions and normalized all ion intensities to the values determined in cells not suffering from hyperosmotic stress, that is, grown in the presence of 50 mM NaCl.…”

“…Because the aqueous extraction procedure used in our metabolomics experiment was not optimal to capture lipids such as Q8, we extracted E. coli cells grown in isosmotic and hyperosmotic conditions with chloroform and methanol 22 and measured lipid levels by flow injection MS 17 . In agreement with our previous metabolomics experiment, non-targeted lipidomics confirmed a 110-fold salt-dependent increase in Q8 levels, the highest fold-change among all detected lipids, concomitant with an apparent decrease of shorter-chain isoprenoid lipids (Fig.…”

“…6). ions were performed using Matlab R2012b (The Mathworks, Nattick, MA, U.S.A.) as described previously 17 .…”

“…Lipids were extracted from exponentially growing E. coli BW25113 cells at OD 600 = 1.0 using a two-step extraction procedure with 17:1 CHCL 3 :methanol (v/v) for non-polar and 2:1 CHCL 3 :methanol (v/v) for polar lipids as described previously 22 . Extracted lipids from 413 mg dry cell weight were vacuum-dried, resuspended in 1:1 acetonitrile:methanol, spiked with fully 13 C-labeled E. coli extract for matrix effect correction and analyzed by flow-injection MS as described previously 17 .…”

“…Specifically, we analyzed the intracellular steady state metabolome of exponentially growing E. coli at six levels of sodium chloride (NaCl)-induced hyperosmotic stress. To this end, we used our previously developed non-targeted flow injection time-of-flight mass spectrometry (MS) platform 17 that allows to measure the semiquantitative levels of hundreds of intracellular metabolites. The acquired data not only shed light on complex metabolic rearrangements, but also led to the discovery of a novel mechanism for coping with osmotic stress, namely the active improvement of cell membrane stability by accumulation of the electron carrier and lipid ubiquinone-8.…”