Abstract
Advances in mass spectrometry have facilitated the identification of novel lipid structures. In this work, we fractionated the lipids of Escherichia coli B and analyzed the fractions using negative-ion electrospray ionization mass spectrometry to reveal unknown lipid structures. Analysis of a fraction eluting with high salt from DEAE cellulose revealed a series of ions not corresponding to any of the known lipids of E. coli. The ions, with m/z 861.5, 875.5, 887.5, 889.5, and 915.5, were analyzed using collision-induced dissociation mass spectrometry (MS/MS) and yielded related fragmentation patterns consistent with a novel diacylated glycerophospholipid. Product ions arising by neutral loss of 216 mass units were observed with all of the unknowns. A corresponding negative product ion was also observed at m/z 215.0. Additional ions at m/z 197.0, 171.0, 146.0, and 128.0 were used to propose the novel structure phosphatidylserylglutamate (PSE). The hypothesized structure was confirmed by comparison with the MS/MS spectrum of a synthetic standard. Normal phase liquid chromatography-mass spectrometry analysis further showed that the endogenous PSE and synthetic PSE eluted with the same retention times. PSE was also observed in the equivalent anion exchange fractions of total lipids extracted from the wild-type E. coli K-12 strain MG1655.
Highlights
Advances in mass spectrometry have facilitated the identification of novel lipid structures
The advent of Electrospray ionization-mass spectrometry (ESI-MS) has confirmed the complexity of the lipidome of E. coli, which was already suspected based on early radiochemical studies [3]
The major ions observed in the direct negative ion ESI-MS analysis of an E. coli total lipid extract, PG, PE, phosphatidic acid (PA), and CL, often obscure the additional minor lipid ions
Summary
Tryptone and yeast extract were from FisherBiotech (Fairlawn, NJ). Glass-backed Silica Gel 60 TLC plates (0.25 mm) and highperformance TLC (HPTLC) plates were from E. The dried lipid film remaining in the tube following solvent removal was redissolved in 2.0 ml of CHCl3:CH3OH (2:1, v/v) This solution was directly infused into the Turbo electrospray ionization source of a QSTAR XL quadrupole time-of-flight tandem mass spectrometer (ABI/MDS-Sciex, Toronto, Canada) at 6 μl/min [15]. The supernatant was transferred to a clean bottle and converted to a two-phase Bligh-Dyer extraction mixture (CHCl3:CH3OH:PBS, 2:2:1.8, v/v/v) by the addition of 62.5 ml of chloroform and 62.5 ml of PBS. The MG1655 total lipid extract was redissolved in 150 ml of CHCl3:CH3OH:H2O (2:3:1, v/v/v) and fractionated on a 15 ml DEAE cellulose column as described above. The fractions that eluted with CHCl3: CH3OH:240 mM NH4Ac (2:3:1, v/v/v) and CHCl3:CH3OH:480 mM NH4Ac (2:3:1, v/v/v) were pooled and converted to a two-phase Bligh-Dyer extraction mixture. Seventy microliters of the sample was analyzed by normal phase LC-MS as described above
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