Qualitative and Quantitative Analysis of Oxidized Fatty Acids by Data Dependent and Data
®
Independent Strategies on a TripleTOF 5600 System
Xu Wang1; Priscilla BMC Derogis2; Sayuri Miyamoto2; Sahana Mollah1; Christie Hunter1
1AB
SCIEX, USA; 2Instituto de Química - Universidade de São Paulo, São Paulo, Brazil
INTRODUCTION
Oxidized fatty acids are a sub class of lipid species derived from the oxidation of poly-unsaturated
fatty acids. These molecules, especially derived from arachidonic acid and docosahexaenoic acid,
collectively known as eicosanoids and docosanoids, are most important lipid autacoids and have
effects on a wide range of physiological and pathophysiological conditions. The challenge of
identifying and quantifying oxidized fatty acids is linked to identical elemental composition and highly
structural similarity. High resolution tandem mass spectrometry using a QqTOF system can contribute
to study of oxidized fatty acids in two ways. Firstly, the accurate mass of characteristic fragment ion
indicates the structural identity of the molecule. Secondly, the intensities of resolved characteristic
fragment ions from isobaric precursors can be used for quantitation. The scope of this work is to
develop and evaluate methods to facilitate future study of oxidized fatty acids, the pathway markers
reflecting the activation of the biosynthesis of specific mediators and inactive products. The
experiments were compared including MRMHR Workflow, IDA, SWATH™ Acquisition, and Infusion
MS/MSALL workflow.
Figure 1. The implementation of
SWATH™
Acquisition
and
Infusion MS/MSALL Workflows.
SWATH Acquisition consists of
one TOF MS experiment and the
consecutive acquisition of 59 TOF
MS/MS experiments across the
chromatographic elution range by
repeatedly stepping through the
mass range of m/z 265-439 (3 Da
Q1 window). MS/MSALL workflow
consists of one TOF MS
experiment and the consecutive
acquisition of 1001 TOF MS/MS
experiments with unit resolution in
Q1 stepping through the mass
rang of m/z 200 -1200.
MATERIALS AND METHODS
Chromatography: Chromatography separation of extracted lipids was performed on a Shimadzu
Prominence UFLC system using ZORBAX Eclipse XDB-C18 column (2.1 mm x 50 mm, 1.8 µm
particle size) (Agilent, Santa Clara, CA). The column oven was operated at 40 ˚C. Mobile phase A
consisted of H2O and 0.05% acetic acid, and mobile phase B consisted of MeOH and 0.05% acetic
acid. An optimized gradient: 55 % B for 2 min, from 55 % B to 85 % B in 8 min, from 85% B to 98 % B
in 8 min, 98% B for 2 min, from 98 % B to 55 % B in 2 min, and 55 % B for 5 min at a flow rate of 0.4
mL/min was used to maximally separate different lipid species. The total run time of each injection
was 27 min. This LC method was applied to all experiments.
Data Processing: PeakView® Software was used for manual data review. Molecular structures used to
confirm the structural identity of oxidized fatty acids were retrieved either automatically by PeakView®
software from ChemSpider (http://www.chemspider.com) based on the accurate precursor ion masses, or
manually from LIPID MAPS (http:www.lipimaps.org). The fragment ion mass was assigned automatically by
Formula Finder in the PeakView® Software and reviewed manually. Batch processing of lipid identification
and quantification was performed by MasterView™ Software. MultiQuant™ Software was used to generate
targeted quantitation methods for relative comparison of selected lipids. These methods can be used for
routine data analysis.
Results
IDA (Information Dependent Acquisition). The experiment was performed on an AB SCIEX
TripleTOF® 5600+ system. Data was acquired in top-35 data-dependent mode: TOF MS for precursor
ion isolation and TOF MS/MS for product ion scan. Precursor ions in the mass range of m/z 250-450
with intensities higher than 250 cps (counts-per-second) were considered for fragmentation. An
inclusion list containing the masses of major oxidized lipids was used to assist identification.
MRMHR Workflow. Multiplexed looped MS/MS acquisition methods targeting on the oxidized fatty
acids of interest were used, with a full scan mass range of m/z 100-1000 and an accumulation time of
50 msec. A full scan TOF MS scan was included I each cycle with an accumulation time of 200 msec.
Q1 isolation window was 0.7 amu. The total cycle time was 1.21 sec.
SWATH™ Acquisition. The SWATH acquisition was performed using Q1 isolation width of 3 amu
across precursor mass range of m/z 265-439 with 59 swath windows (Figure 1). The total cycle time
was 1.28 sec with TOF MS accumulation time of 50 msec and MS/MS accumulation time of 20 msec.
Automated Acquisition with Infusion MS/MSALL Workflow. Automated infusion was performed on
an ekspert™ microLC 200 system (Eksigent, USA) system. Mobile phases that consisted of
CH2Cl2/MeOH (50/50 v/v) and 2 mM NH4OAc was flowing at 4 µL/min and lasted 5.1 min for data
acquisition. A 25 µm ID hybrid electrode (Eksigent) was used in the DuoSpray™ Source for stable
spray and reduced peak dispersion. The acquisition was controlled by Analyst® TF 1.6.1 software with
MS/MSALL mode activated. First, the TOF MS is scanned from m/z 100-1500. Then 1001 MS/MS
spectra are acquired sequentially from precursor m/z 200.050 to 1200.051, stepping at 1 amu
intervals (Q1 at a 1 Da isolation width) (Figure 1). Each MS/MS spectrum covered the mass range of
100-1500 as well.
Identification of Oxidized Fatty Acids
We performed experiments on a tandem mass spectrometry that consists of full spectrum of lipid precursors
(MS) following by MS/MS experiments to acquire fragment ion mass. The accurate precursor mass was used
to obtain the formula composition of intact lipid, and MS/MS provided additional information for the underlying
lipid-specific characteristic fragment ions to confirm the identification and/or distinguish isobaric species.
Because of the isobaric properties of oxidized lipids, the characteristic fragment ions are required for
identification (Figure 2). Identified oxidized fatty acids from IDA, MS/MSALL, and SWATH are summarized in
Table 1.
Figure 2. High Resolution MS/MS of
5HETE and 11HETE Acquired in the
SWATH Acquisition Experiment. Most
of the fragment ions were shared
between the isoforms. The highlighted
characteristic fragments on the structure
were used for identification.
Table 1. Identified oxidized fatty acids with their retention times and characteristic fragments.
Name
Precursor*
Fragment*
RT
IDA
MS/MSALL
5HEPE
11HEPE
12HEPE
15HEPE
9HEPE
12-oxo-ETE
5-oxo-ETE
15-oxo-ETE
11-oxo-ETE
5HETE
11HETE
12HETE
13HETE
15HETE
20HETE
8HETE
9HETE
18HETE
14,15EpETrE
5,6EpETrE
8,9EpETrE
11,12EpETrE
RvE2
15HpEPE
Hepoxilin A3
Hepoxilin B3
5,15HETE
5,6HETE
LTB4
15-HpETE
14.15-DiHETrE
8.9-DiHETrE
4HDHA
7HDHA
10HDHA
14HDHA
17HDHA
11HDHA
13HDHA
16HDHA
20HDHA
RvD5 (7,17)
RvD6 (4,17)
PD1
20-carboxy-LTB4
317.2122
317.2122
317.2122
317.2122
317.2122
317.2122
317.2122
317.2122
317.2122
319.2279
319.2279
319.2279
319.2279
319.2279
319.2279
319.2279
319.2279
319.2279
319.2279
319.2279
319.2279
319.2279
333.2071
333.2071
335.2228
335.2228
335.2228
335.2228
335.2228
335.2228
337.2384
337.2384
343.2279
343.2279
343.2279
343.2279
343.2279
343.2279
343.2279
343.2279
343.2279
359.2228
359.2228
359.2228
365.1970
201.1650
167.1071
179.1071
219.1384
149.1337
153.1285
203.1805
113.0966
123.1169
115.0394
167.1072
153.1280
193.1229
175.1492
289.2168
155.0707
151.1488
289.1810
113.0966
83.0496
127.1122
163.0759
271.2068
111.0445
195.1021
183.1021
115.0394
219.1755
151.1128
113.0965
207.1384
127.0758
101.0238
141.0551
181.0864
205.1235
245.1548
165.0915
193.1227
233.1540
285.1861
199.1494
256.1833
153.0915
195.1020
9.47
9.64
9.44
9.50
9.78
9.72
9.97
9.68
9.65
10.05
9.66
9.72
9.46
9.52
9.76
9.76
9.91
8.44
9.51
10.01
9.44
9.72
10.98
9.74
8.50
8.50
10.04
9.84
9.43
9.50
10.56
10.99
10.34
9.95
9.81
9.81
9.67
9.93
9.73
9.62
9.53
9.54
9.86
9.60
8.34
N
Y
Y
Y
N
Y
Y
Y
N
Y
Y
Y
Y
Y
N
Y
Y
N
Y
Y
Y
Y
Y
N
Y
Y
N
Y
Y
Y
N
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
N
N
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
N
Y
Y
N
Y
Y
Y
Y
Y
Y
Y
N
N
Y
Y
Y
N
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
N
Y
N
Quantitative Evaluation of
Oxidized Lipids
The combination of LC resolving power and
accurate precursor ion mass has been helpful to
quantitate lipid species from complex sample.
However, this approach cannot be used for
quantitation of oxidized fatty acids due to the
isobaric overlap of precursors (Figure 3). In this
case, the characteristic fragment ion in the
MS/MS becomes an essential factor for oxidized
fatty acids quantitation.
MS/MS spectra were acquired in both SWATH™
Acquisition and MS/MSALL experiments. Figure 4
showed comparable results between the two
methods. It highlights that MS/MSALL was
capable of quantifying oxidized fatty acids.
Although MS/MS spectra were also acquired in
the IDA experiment, they cannot be used to
generate XIC spectra for quantitation due to the
stochastic samplingof a given precursor.
SWATH
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
HEPE,
hyroxy-eicosapentaenoic
acid; oxo-ETE, oxo-eicosatetraenoic
acid;
HETE,
hydroxyeicosatetraenoic acid; EpETrE,
epoxy-eicosatrienoic
acid;
RvE, trihydroxy-eicosapentaenoic
acid;
HpEPE,
hydroperoxyeicosapentaenoic
acid;
LTB4,
dihydroxy-eicosatetraenoic
acid;
HpETE,
hydroperoxyeicosatetraenoic acid ; DiHETrE,
dihydroxy-eicosatrienoic
acid
;
HDHA, hydroxy-docosahexaenoic
acid
;
RvD,
trihydroxydocosahexaenoic acid, carboxyLTB4, dihydroxy-eicosatetraene-1,
20-dioic acid ; PD1, protectin D1
Figure 3. HETE Isoforms. XIC of 319.2279 for HETE
isoforms in TOF MS (top) and XIC of selected HETE
isoforms using characteristic fragment ions (bottom).
On the other hand, we performed a targeted quantitation experiment built by looping a series of product
ion scans based on the list of identified oxidized lipids, so called MRMHR experiment. MRMHR runs similar
to SWATH but targets on precursor. The results showed MRMHR improved the specificity for easy data
analysis (Figure 5). Overall, MRMHR results showed identical quantitation results as SWATH (Figure 4).
Figure 4. The Relative Quantitation Profile of
Selected HDHA and HETE. For a better comparison,
the intensity (bar height) of each lipid was represented
by a normalized value to compare between the
integrated XIC peak area from SWATH™ Acquisition
and MRMHR Workflow and the fragment peak height
from infusion MS/MSALL. Overall, it shows relatively
comparable results from three methods. Low abundant
lipids including 20 HETE and 18 HETE were only
quantifiable in SWATH data.
Figure 5.
Extracted Ion Chromatograms of
Selected HDHAs from SWATH™ Acquisition and
MRMHR Workflow. A and C are XICs of 343.2279
from SWATH™ and MRMHR showing identical
elution profile of HDHA isoforms. B and D are and
XICs of selected HDHA isoforms using characteristic
fragment ions. The isobaric interferences, as shown
in D, were observed from SWATH™ result due to
the wider isolation window.
SWATH
MS/MSALL
MRMHR
CONCLUSIONS
• Both infusion MS/MSALL and SWATH™ Acquisition were DIA experiments, in which a complete record
of all detectable precursor ions and their product ions were collected. They are capable of quantifying all
detectable oxidized fatty acids using characteristic fragment ion.
• SWATH™ Acquisition identified more oxidized fatty acids than infusion MS/MSALL because of the
benefits of LC/MS for reducing ion suppression. However the throughput of the MS/MSALL method was
about 4 fold higher than SWATH™.
• IDA experiment can be used to identify oxidized fatty acids, but it cannot be used for quantify oxidized
fatty acids using characteristic fragment ions due to the intermittent acquisition of a given precursor.
• MRMHR Workflow provides both the advantage of LC and improved specificity with simplified data
analysis, when targeted a specific set of lipids is required.
TRADEMARKS/LICENSING
For Research Use Only. Not for use in diagnostic procedures.
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AB SCIEX™ is being used under license.
© 2014 AB SCIEX.