Quantification of glycerophospholipids in Escherichia coli

Marquise L. Hopson, ‘10 and Teresa A. Garrett, Ph. D, Department of Chemistry, Vassar College
Introduction Methods Results
Phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglyc-
13 Supernatant to fresh U1
erol (PG), and cardiolipin (CL) are the the major classes of lipids produced in tube
+ L1
stds
Escherichia coli. Each class contains numerous possible molecular species
that include multiple compounds of the same molecular formula. For ex- Single phase Bligh-Dyer
1 CHCl 3 : 2 CH 3 OH: .8 PBS
Two phase Bligh-Dyer
1 CHCl 3 : 2 CH 3 OH: .8 PBS
ample, the species PA 32:1 includes any PA compound that has 32 total car-
bons in the acyl chains and one unsaturation, including PA 14:0/18:1, PA
16:0/16:1, etc. The goal of this project is to quantify the total amount of PA, Wash lower
Remove upper
Dry with N2
L1 phase
PE, PG, and CL as well as the amount of each species, as defined by the total
phase , mix gas

number of carbons and unsaturations in the acyl chains, within each class in
E. coli cells.
O
Figure 3 Wildtype E. coli were grown to log phase and the Bligh-Dyer method was used
HO O
to extract lipids from the cells. Thirteen lipid standards were also coextracted with the
O O
P
Compound- PA 16:0/ 16:1 cells. Dried lipid samples were sent to Duke University for LC-ESI-MS analysis.
OH
O Species- PA 32:1
O
Data Analysis
O HO O
From the resulting spectra, peak areas for the extracted ion current
P
Compound- PA 14:0/ 18:1 were recorded for each endogenous species and standard
O O
OH Species- PA 32:1
O

Total ion current

O

Figure 1 Both PA 16:0/ 16:1 and PA 14:0/ 18:1 are the species 32:1, since they have the same Figure 6 Endogenous species abundance in E. coli cells. Clockwise from the upper left corner: PE, PA, CL, and PG. Eighty
total number of carbons and double bonds in the acyl chains. Compounds that are the same total endogenous species and standards were calculated.
species have the same molecular formula and exact mass. Extracted ion current

Liquid chromatography electrospray ionization mass spectrometry (LC-ESI-

MS) is used to quantify the lipids in this experiment. LC-ESI-MS separates
each compound by polarity and mass-to-charge ratio. The four classes stud- Time of flight mass spectrum PE
79%
ied in this experiment elute off of the liqud chromatography column at dif-
ferent times. The instrument detects the compounds and displays the total
ion current, a measure of intensity vs. time, from which we can isolate spe-
cific ions to view the extracted ion current, a spectrum of intensity vs. mass-
Figure 4 Example spectra from LC-ESI-MS. The total ion current (TIC), which includes all PG
to-charge ratio. The integrated peak areas from the spectra of intensity vs. endogenous species and standards. The extracted ion current (XIC) shown is from TIC of 19%
time for each are used to calculate the abundance in cells. The purpose of compounds 645-646 m/z. The time of flight mass spectrum (TOF MS) of the extracted
this experiment is to use the data obtained using LC-ESI-MS to calculate the ion shows which ions elute at a particular time. PA
CL
percent compostion of PE, PA, PG, and CL in E. coli. 1%
0.2%

A Peak Area Analyte

X ng Standard Added = ng analyte
Peak Area Standard

sample LC column Ionization Figure 7 The amount of each species was summed to calculate the percent composition of PA, PE, PG, and CL in E. coli cells.
solution B

1ng ? The results are consistent with previous literature from Raetz, et al. 1979, who
observed PE 82.1%, PG 16.2%, and CL 1.6%.

Detection M/z separation

Future Work

Resolve issues with PE standard curves that were used to calculate total amount
of analyte
Figure 2 A model of liquid chromatography electrospray ionization mass spectrometry (LC-
ESI-MS), a highly selective method that separates each compound by polarity, ionizes the
molecules, and then further separates the compounds by mass-to-charge ratio. Mass spec-
Figure 5 (A) Equation used to calculate the amount of each species in cells. (B) Ex-
Vary conditions of the experiment, such as using different standards or growing
trometry separates the compounds by mass, thus pecies, such as PA 32:1, can be identified
but the specific composition of the acyl chains is not known.
ample standard peak and 32:1 species peak. The recorded information about the stan- cells at different temperatures and pH levels
dard, 1ng added and peak area = 100, and the 32:1 species, peak area = 75, is used to
calculate the amount of 32:1 present in the samples.