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SUPPLEMENTAL INFORMATION FOR
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Deuterium-exchange Metabolomics Reveals N-Methyl
lyso Phosphatidylethanolamines As Abundant Lipids in
Acidophilic Mixed Microbial Communities
Curt R. Fischer1*, Paul Wilmes1,5*, Benjamin P. Bowen3,4, Trent Northen3,4, Jillian F.
Banfield1,2
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1.1 Metabolite extraction and LC-ESI-qTOF-MS analysis.
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Supplemental Methods
Natural biofilm samples. Approximately 500 mg of frozen biomass (-80 oC) was
removed from each sample and analyzed by LC-ESI-qTOF-MS as described
previously [3]. Cells were pelleted by centrifugation (4,000 rpm) and resuspended
in an equivalent volume of ice cold 1 x PBS buffer at pH 1 to avoid inconsistent cell
lysis prior to sonication. The pH of the 1 x PBS buffer was adjusted using a 5 M
H2SO4 solution. Micro-tip sonication was performed on ice for four 30 s bursts. This
was followed by the addition of 1,000 μL of hot methanol (65 oC), vortexing and
incubation at 65 oC for 3 min. The biomass was pelleted by centrifugation (14,000
rpm), 750 μL of supernatant were removed and the pellets re-extracted with 500 μL
of hot isopropanol (65 oC) for 3 min. The biomass was again pelleted by
centrifugation, the extracts pooled, dried by vacuum centrifugation, redissolved in a
1:2:2 mixture of water:methanol:IPA (100 μL/mg of wet biomass), and filtered (0.45
mm PVDF, National Scientific, Rockwood, TN, USA).
Bioreactor biofilm samples. Frozen 250 mg to 500 mg of -80 °C conserved bioreactor
biofilm pellets were removed from bulk frozen samples and weighed. One volume
(w/v) of PBS and two volumes of methanol were added to each biofilm sample. The
sample was sonicated on ice at high power (~200 W) for 5 min using a microtipequipped Sonicator 350 (Heat Systems Ultrasonics, Farmingdale, NY). Frequent
pauses were inserted in each sonication cycle to avoid overheating. After sonication,
samples were vortexed, and centrifuged for 10 min at 10,000 g. Two volumes of
methanol/PBS-rich supernatant were removed and transferred to a separate
container. Two volumes of isopropanol were added to the sonicated lysate , and the
sample was vortexted and re-sonicated. The isopropanol extract was centrifuged,
and two volumes of supernatant were removed from above the cell debris and
added to the separate container containing MeOH/PBS supernatant.
These mixed supernatants were dried by vacuum centrifugation, and stored at 20 °C. Prior to LC-MS analysis, stored dried extracts were reconstituted in 100 uL of
2:2:1 (v:v:v) methanol:isopropanol:water and filtered through 0.22 um PVDF
microfilters prior to LC-MS analysis.
Reverse phase chromatography was performed using 4 μL of extract injected onto a
150 × 0.5 mm Zorbax C-18 column (Agilent Technologies, Santa Clara, CA, USA) with
5 mm particles at a flow rate of 20 L/min. Buffer A was HPLC water with either
0.1% formic acid or 0.1% acetic acid, and buffer B was acetonitrile with either 0.1%
formic acid or 0.1% acetic acid. Chromatography was performed as follows: the
column was equilibrated in 3% buffer B, held for 3 min, and then the gradient was 3
- 50% buffer B over 5 min, 50 - 99% buffer B over 25 min, held at 99% for 10 min,
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and then re-equilibrated in 3% buffer B for 10 min. Sample run order was
randomized to control for carry-over and instrument drift.
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1.2 Data visualization in Kendrick mass defect diagrams
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The Kendrick mass or Kendrick m/z ratio is the mass or m/z value in Daltons
multiplied by 14.000000 / 14.015650. On this mass scale, CH2 units have an integer
mass. A mass defect diagram plots the integer mass of a feature vs. the mass “defect”
or decimal part of the mass [1]. Addition or subtraction of CH2 units (e.g., by
replacement of a hydrogen with a methyl group) will result in purely horizontal
shifts on a Kendrick mass defect diagram. Addition or subtraction of other moieties
will result in diagonal shifts.
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1.3 Chemical standards.
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Authentic standards of ethanolamine, N-methyl ethanolamine, and N,N dimethyl
ethanolamine were purchased from Sigma-Aldrich. Authentic standards of 1palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-snglycero-3-phosphoethanolamine, and 1-stearoyl-2-Hydroxy-sn-Glycero-3phosphoethanolamine were purchased from Avanti Polar Lipids, Inc.
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1.4 Estimation of absolute abundance for lyso phospholipids and comparison to
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Positive mode data was collected over a range of m/z 50-2000 using an Agilent
Technologies (Santa Clara, CA, USA) ESI-qTOF Model 6520 (typical resolution 14k)
with a capillary voltage of 4,000 V, a drying gas flow rate of 3.5 L/min, scan rate of
1.03s/spectra, and a nebulizer gas pressure of 10 psi. Each raw data file, was
converted to mzXML using Trapper (Agilent Technologies).
diacyl lipid abundance
Absolute quantification of the lyso lipids in bioreactor-derived AMD biofilm was
made by spiking between 7.6 and 253 nmol of 18:0 lyso PE (Avanti Polar Lipids) into
lyophilized biomass samples before extraction. To ensure complete lipid extraction,
a lipid-specific extraction protocol was used: biomass samples were freeze-dried
overnight (Labconco FreeZone2.5, Labconco, Kansas City, MO) and extracted with a
2:1 methanol:chloroform solvent system. The resulting extracts were briefly
disrupted by agitation with a steel ball (Biospec Products Mini-beadbeater 96,
Biospec Products, Bartlesville, OK), vortexing, and low-power sonication (Branson
Sonifier 250, Branson Ultrasonics, Danbury, CT). Extracts clarified after
centrifugation were centrifuged under vacuum and resuspended in 2:2:1
isopropanol:methanol:water and analyzed by LC-MS as described above.
The 18:0 lyso PE lipid is isobaric with the methylated 17:0 lipid naturally found in
the sample and elutes at a very similar chromatographic retention time. It
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fragments by CID in analogous manner to the methylated 17:0 lipid, but fragment
ions have distinct m/z values because the C18:0 lipid has a longer fatty acid chain
and no N-methylation. For example, the natural methylated 17:0 lipid spectrum has
a prominent peak at 327 Da, while the 18:0 lyso PE standard has an internal has a
peak at 341 Da. Across the range in internal standard loadings used (between 7.6
and 253 nmol), instrument response was approximately linear. Absolute
abundance of the 482 lipid was estimated from the peak intensity ratio of the
internal standard and the sample-derived methylated 17:0 lipid and knowledge of
the absolute amount of internal standard loaded.
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1.5 Data processing.
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For metabolomics profiling, data for each sample were converted to mzXML format
and analyzed using the software xcms [2], which provides peak-finding, integration,
and feature grouping functionalities. Features co-eluting with the solvent front or a
very intense, highly complex envelope of features at 500 seconds were discarded to
avoid the complexities of ion suppression and mass red-shifting; only features with
retention times of > 600 seconds were retained for further analysis.
For 15N- or proton-painting experiments, MassHunter software (Agilent
Technologies, Santa Clara, CA) was used to extract ion chromatograms (at +/- 5 ppm
mass tolerance) at m/z values predicted from the m/z value of the unlabeled
monoisotopic mass of the target feature and the number of label-able atoms in the
target formula.
Hierarchical clustering (Pearson uncentered) of unit vector normalized PE and
MMPE MS1 signal intensities was carried out in the MultiExperiment Viewer
package (Linkage method: average linkage; leaf order optimization: none;
http://www.tm4.org/mev/).
Strained normalized spectral abundance factor (S-NSAF) values were used as the
metric for protein abundance and unit vector normalized metabolite feature
intensities were used as the metric for metabolite feature abundance, as decribed
previously [3]. In order to avoid potential biases in the data arising from zero values,
we focused our analyses on protein and metabolite features that were detected in all
of the sampled communities. The R programming language was used to calculate the
Pearson product-moment correlation coefficients (r; cor function) and
corresponding p-values (cor.pvals function) for all possible protein-metabolite
feature pairs. Correlated features and their associated r- and p-values were
extracted using custom Ruby scripts (available from the authors upon request). Only
r-values with an associated statistical p-value of ≤ 0.0001 were considered. To
achieve a balanced analysis, an r value was chosen that relates an equal number of
proteins and metabolite features [36]. Regression analysis revealed an equivalent
sampling of proteins and metabolite features at r ≥ 0.9285 (n = 370).
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Correlation networks at r ≥ 0.9285 for correlated protein-metabolite features,
proteins and metabolite features were constructed in Cytoscape version 2.6.3 using
pairs of correlated proteins and/or metabolite features (nodes) with their
corresponding r (edges) as input and using the circular layout function in the y-files
plugin for Cytoscape.
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Mass spectra of authentic ethanolamine standards and MS2
fragments of detected lipids
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Absolute quantification
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Proton painting data for all lipids and example spectra of mixed
isomers for m/z = 466 and m/z = 468 features.
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Blast information for putative pmtA genes
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Table S1. Blast summary statistics for AMD genes with high homology to known
phosphatidylethanolamine N-methyl transferase genes.
ORF
Best hit vs. K00551
UBA_LeptoII_Scaf_8524_GENE_228
5wayCG_LeptoII_Cont_11276_GENE_62
UBA_LeptoIII_Cont_9568_GENE_79
ORF
ava:Ava_2605
ava:Ava_2605
ava:Ava_2605
Best hit vs. nr
A. manganoxydans S185-9A1
UBA_LeptoII_Scaf_8524_GENE_228
methyltransferase, EAS49908
A. manganoxydans S185-9A1
5wayCG_LeptoII_Cont_11276_GENE_62
methyltransferase, EAS49908
S. viridis DSM43017 methylase in
UBA_LeptoIII_Cont_9568_GENE_79
quinone biosynthesis
YP_003132138.1
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K00551 best-hit
Eval
9.00E-23
7.00E-23
3.00E-26
nr best hit Eval
1.00E-21
4.00E-22
3.00E-25
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Correlation analysis of combined metabolomic and proteomic data
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Supplemental References
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[1]
C.A. Hughey, C.L. Hendrickson, R.P. Rodgers, A.G. Marshall, and K. Qian.
Kendrick mass defect spectrum: a compact visual analysis for ultrahigh-resolution
broadband mass spectra. Anal. Chem, 73(19):4676–4681, 2001.
[2]
Colin A Smith, Elizabeth J Want, Grace O'Maille, Ruben Abagyan, and Gary
Siuzdak. Xcms: processing mass spectrometry data for metabolite profiling using
nonlinear peak alignment, matching, and identification. Anal Chem, 78(3):779–787,
Feb 2006.
[3]
P. Wilmes, B.P. Bowen, B.C. Thomas, R.S. Mueller, V.J. Denef, N.C.
VerBerkmoes, R.L. Hettich, T.R. Northen, and J.F. Banfield. Metabolome-Proteome
Differentiation Coupled to Microbial Divergence. mBio, 1(5):e00246, 2010.
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