H
OH
OH
metabolites
Article
Monoacylglycerol Analysis Using MS/MSALL
Quadruple Time of Flight Mass Spectrometry
Fei Gao 1 , Justice McDaniel 1 , Emily Y. Chen 1 , Hannah Rockwell 1 , Matthew D. Lynes 2 ,
Yu-Hua Tseng 2 , Rangaprasad Sarangarajan 1 , Niven R. Narain 1 and Michael A. Kiebish 1, *
1
2
*
BERG, LLC, 500 Old Connecticut Path, Bldg B, 3rd Floor, Framingham, MA 01701, USA;
[email protected] (F.G.); [email protected] (J.M.);
[email protected] (E.Y.C.); [email protected] (H.R.);
[email protected] (R.S.); [email protected] (N.R.N.)
Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School,
Boston, MA 02215, USA; [email protected] (M.D.L.);
[email protected] (Y.-H.T.)
Correspondence: [email protected]
Academic Editors: Vladimir Tolstikov and Peter Meikle
Received: 16 June 2016; Accepted: 12 August 2016; Published: 17 August 2016
Abstract: Monoacylglycerols (MAGs) are structural and bioactive metabolites critical for biological
function. Development of facile tools for measuring MAG are essential to understand its role in
different diseases and various pathways. A data-independent acquisition method, MS/MSALL ,
using electrospray ionization (ESI) coupled quadrupole time of flight mass spectrometry (MS), was
utilized for the structural identification and quantitative analysis of individual MAG molecular
species. Compared with other acylglycerols, diacylglycerols (DAG) and triacylglycerols (TAG), MAG
characteristically presented as a dominant protonated ion, [M + H]+ , and under low collision energy
as fatty acid-like fragments due to the neutral loss of the glycerol head group. At low concentrations
(<10 pmol/µL), where lipid-lipid interactions are rare, there was a strong linear correlation between
ion abundance and MAG concentration. Moreover, using the MS/MSALL method the major MAG
species from human plasma and mouse brown and white adipose tissues were quantified in less
than 6 min. Collectively, these results demonstrate that MS/MSALL analysis of MAG is an enabling
strategy for the direct identification and quantitative analysis of low level MAG species from biological
samples with high throughput and sensitivity.
Keywords: monoacyglycerols; MS/MSALL ; mass spectrometry; QTOF; acylglycerols
1. Introduction
Monoacylglycerol (MAG) is a glyceride in which a glycerol molecule forms an ester bond with
exactly one fatty acid (FA) molecule in either sn-1 or sn-2 positions. MAG is either biosynthesized
as an intermediate during lipogenesis or formed by the hydrolysis of fat (mainly of triacylglycerols)
(Figure 1). MAG plays an important role in lipid metabolism and homeostasis, as it is positioned in
the center of the lipid pathways and controls the flow and distribution of fatty acids to other complex
lipids, such as triacylyglycerols (TAG) and phospholipids [1]. The level of MAG in any biological
system is tightly regulated by several enzymes, in particular monoacylglycerol lipase (MAGL). Notably,
the disruption of MAGL can perturb the balance of monoglycerides, including the endocannabinoid
2-arachidonoyl [2,3], and even promote cancer pathogenesis [4].
Metabolites 2016, 6, 25; doi:10.3390/metabo6030025
www.mdpi.com/journal/metabolites
the analysis of MAG is carried out using gas chromatography (GC) and gas chromatography mass
spectrometry (GC-MS) [5–7] after derivatization, or reversed-phase HPLC with a UV detector [8].
However, separation and derivatization is time-consuming, and thus does not meet high throughput
requirements for application using clinical samples. Moreover, the complexity of matrix and lipid species
further
requires
Metabolites
2016, 6, 25tailored analytical techniques to accurately quantify MAG species in various sample
2 of 13
types.
Figure
pathways
of monoacylglycerol
(MAG). LPL,
lipoprotein
lipase; DGAT,
diacylglycerol
Figure1.1.Biological
Biological
pathways
of monoacylglycerol
(MAG).
LPL, lipoprotein
lipase;
DGAT,
acyltransferase;
MGAT,
monoacylglycerol
acyltransferase;
MAGL,
monoacylglycerol
lipase;
DAG,
diacylglycerol acyltransferase; MGAT, monoacylglycerol acyltransferase; MAGL, monoacylglycerol
diacylglycerol;
TAG,
triacylglycerol;
LPA,
lysophosphatidyl
acid;
FA,
fatty
acid.
lipase; DAG, diacylglycerol; TAG, triacylglycerol; LPA, lysophosphatidyl acid; FA, fatty acid.
Due to recent advances in mass spectrometry and ionization technology, the molecular species of
MAGs are mostly found at very low levels in human plasma and organ tissues. As such,
lipids can be analyzed by electrospray ionization-mass spectrometry (ESI-MS) coupled with HPLC
quantitative analysis is a considerable challenge
as it requires highly sensitive methods for
separation [9,10]. MAG usually forms [M + H]+ or adduct ions, which can be further fragmented into fatty
detection. Traditionally, the analysis of MAG is carried out using gas chromatography (GC) and
acid related ions for structural elucidation. Under a tandem mass spectrometry strategy, lipid classes,
gas chromatography mass spectrometry (GC-MS) [5–7] after derivatization, or reversed-phase HPLC
such as glycerophospholipids, glycerolipids, and sphingolipids, can be identified based on their
with a UV detector [8]. However, separation and derivatization is time-consuming, and thus does not
characteristic fragmented ion. Traditionally, these types of ions have been used in precursor ion scanning,
meet high
requirements
for application
usingexperiments,
clinical samples.
Moreover,
complexity
neutral
lossthroughput
scanning, and
multiple reaction
monitoring
to measure
the the
lipid
molecular
of
matrix
and
lipid
species
further
requires
tailored
analytical
techniques
to
accurately
quantify
species, coupled with either direct infusion, e.g., shotgun lipidomics, or liquid chromatographyMAG
(LC)
species
in
various
sample
types.
modes, mainly on triple quadrupole (QQQ) instruments [11,12]. However, the drawbacks of such
Dueare
to recent
advances
in mass
spectrometry
ionization
technology,ofthe
molecular
of
analysis
that they
are highly
targeted
and they and
require
the pre-selection
specific
ions. species
Currently,
lipids
can
be
analyzed
by
electrospray
ionization-mass
spectrometry
(ESI-MS)
coupled
with
HPLC
there are two alternative strategies for global lipid determination: data-dependent acquisition (DDA) and
separation [9,10]. MAG
usually
forms
[M is
+ typically
H]+ or adduct
which can
further
fragmented
into
data-independent
acquisition
(DIA).
DDA
basedions,
on surveying
anbe
MS
scan and
involves userfatty
acid
related
ions
for
structural
elucidation.
Under
a
tandem
mass
spectrometry
strategy,
lipid
defined information dependent criteria to select the precursor ions for MS/MS fragmentation. DDA
classes,the
such
as glycerophospholipids,
glycerolipids,
and sphingolipids,
be identified
based
on
allows
identification
of unknown lipid
molecular species
and is usually can
coupled
with an LC
system.
their characteristic
fragmented
ion. Traditionally,
typeslimitations,
of ions have
been
used
in precursor
However,
the analysis
of global lipidomics
using DDAthese
has some
such
as poor
reproducibility,
ion scanning,
neutral
and multiple
reactionlipid
monitoring
experiments,
to measurethese
the
limited
dynamic
range,loss
andscanning,
bias towards
high abundance
species [13–15].
DIA overcomes
lipid molecular
species, coupled
with either
direct
infusion,
shotgun
lipidomics,
liquid
limitations
by parallelizing
the fragmentation
of all
detectable
ionse.g.,
within
a certain
m/z range, or
providing
modes,
on triple and
quadrupole
(QQQ) instruments
[11,12]. However,
achromatography
broader dynamic(LC)
range,
highermainly
reproducibility,
better sensitivity
[13].
the drawbacks of such analysis are that they are highly targeted and they require the pre-selection
of specific ions. Currently, there are two alternative strategies for global lipid determination:
data-dependent acquisition (DDA) and data-independent acquisition (DIA). DDA is typically based on
surveying an MS scan and involves user-defined information dependent criteria to select the precursor
ions for MS/MS fragmentation. DDA allows the identification of unknown lipid molecular species and
is usually coupled with an LC system. However, the analysis of global lipidomics using DDA has some
limitations, such as poor reproducibility, limited dynamic range, and bias towards high abundance lipid
species [13–15]. DIA overcomes these limitations by parallelizing the fragmentation of all detectable
ions within a certain m/z range, providing a broader dynamic range, higher reproducibility, and better
sensitivity [13].
Herein, we report a high throughput and highly sensitive MS/MSALL workflow for profiling
MAG molecular species via the direct infusion of lipid extracts on a quadrupole time of flight mass
Metabolites 2016, 6, 25
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6, 25
3 ofglobal
14
spectrometer
[13,16].
MS/MSALL is a direct infusion DIA technique, specifically designed for
lipidomics [13,16]. In this report, all precursor ions in the Q1 quadrupole
at
1
Da
m/z
window
Herein, we report a high throughput and highly sensitive MS/MSALL workflow for profiling MAG
were selected. MS/MS fragmentation was then carried out in Q2, and TOF collected all the high
molecular species via the direct infusion of lipid extracts on a quadrupole time of flight mass
resolution
MS/MS
spectra
at aALLhigh
These conditions
and parameters
also be
is aspeed.
direct infusion
DIA technique,
specifically can
designed
forperformed
global
spectrometer
[13,16].
MS/MS
on lipidomics
other Q-TOF
or
hybrid
orbitrap
mass
spectrometers
with
the
correct
software
and
[13,16]. In this report, all precursor ions in the Q1 quadrupole at 1 Da m/z windowhardware
were
parameters,
which fragmentation
thus allows for
broad
here.
For MAG
species,
selected. MS/MS
was
then utilization
carried out of
in the
Q2, method
and TOFdescribed
collected all
the high
resolution
spectra
at afragmentation
high speed. These
conditionswere
and parameters
also be
performed
onaother
Q-TOFand
the MS/MS
ionization
and
conditions
optimizedcan
using
17:1
MAG as
standard
or hybrid
mass spectrometers
the correct software
andtriacylglycerol
hardware parameters,
thus
compared
to orbitrap
two common
acylglycerols,with
diacylglycerol
(DAG) and
(TAG).which
The approach
allows
for
broad
utilization
of
the
method
described
here.
For
MAG
species,
the
ionization
presented here demonstrates robust quantification of low level MAGs in human plasma andand
mouse
fragmentation
were optimized
using
17:1 MAG
a standard
andrange,
compared
tothroughput,
two common and
brown
and whiteconditions
adipose tissues
in less than
6 min
with aaswide
dynamic
high
acylglycerols, diacylglycerol (DAG) and triacylglycerol (TAG). The approach presented here
high sensitivity.
demonstrates robust quantification of low level MAGs in human plasma and mouse brown and white
adipose tissues in less than 6 min with a wide dynamic range, high throughput, and high sensitivity.
2. Results
2. Results
2.1. Characterization MAG, DAG, and TAG Ionization Efficiency
2.1.
MAG, DAG,
and TAG Ionization
ToCharacterization
optimize the ESI-MS
and MS/MS
ionizationEfficiency
efficiency of MAG compared to other acylglycerols
ALL
DAG and
using
a mixtureefficiency
of equalofmolar
(1 µM)
of MAG 17:1,
ToTAG
optimize
theMS/MS
ESI-MS andanalysis,
MS/MS ionization
MAGconcentration
compared to other
acylglycerols
ALL analysis,
DAG
d17:1,
and TAG
was
infused ainto
the mass
spectrometer
to optimize
compare
mixture
of equal
molar concentration
(1 µM)and
of MAG
17:1, the
DAG
and TAG
using t17:1
MS/MS
DAG d17:1,
TAG t17:1 was
infusedofinto
the three
mass spectrometer
to optimize
and compare
the as
ionization
andand
fragmentation
patterns
these
acylglycerides.
Non-polar
lipids such
ionization
and
fragmentation
patterns
of
these
three
acylglycerides.
Non-polar
lipids
such
as
acylglycerides easily form adduct ions, so 2 mM ammonium acetate was added into the mobile phase
acylglycerides
easily
form
adduct
ions,
so
2
mM
ammonium
acetate
was
added
into
the
mobile
phase
(isopropanol:methanol:acetonitrile:H2 O (3:3:3:1, by vol.)), to facilitate the formation of ammonium
(isopropanol:methanol:acetonitrile:H2O (3:3:3:1, by vol.)), to facilitate the formation of ammonium
adduct ions [M + NH4 ]++ . Under the experimental conditions, as expected, ammonium adducts of
adduct ions [M + NH4] . Under the experimental conditions, as expected, ammonium adducts of DAG
DAG d17:1 and TAG t17:1 appeared as dominant molecular ions, whereas the ionization of MAG 17:1
d17:1 and TAG t17:1 appeared as dominant molecular ions, whereas the ionization of MAG 17:1 showed
showed
two offorms
of molecular
ions
with
the major
ionthe
atminor
m/z ammonium
343.2 and the
minor
two forms
molecular
ions with the
major
protonated
ion protonated
at m/z 343.2 and
adduct
ammonium
ion at2).m/z 360.3 (Figure 2).
ion at m/z adduct
360.3 (Figure
Figure
2. ESI-MS
spectrafor
forequal
equal molar
molar concentration
17:1,
DAG
d17:1,
andand
TAGTAG
t17:1.
Figure
2. ESI-MS
spectra
concentration(1(1µM)
µM)ofofMAG
MAG
17:1,
DAG
d17:1,
t17:1.
Spectra
were
acquired
with
a
TripleTOF
5600+
under
the
following
conditions:
GS1
=
10,
GS2
=
10,
CUR
Spectra were acquired with a TripleTOF 5600+ under the following conditions: GS1 = 10, GS2 == 10,
15, Temp = 300 °C.
CUR = 15, Temp = 300 ◦ C.
In order to yield stable and highly sensitive MS and tandem MS signals for MAG and other
acylglycerols the QTOF parameters were optimized. The declustering potential (DP) is a voltage
applied to the orifice that helps prevent ions from clustering, but high DP can introduce the in-source
fragmentation [17]. Thus, the DP value affects the ionization efficiency and sensitivity, which thus
Metabolites 2016, 6, 25
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In order to yield stable and highly sensitive MS and tandem MS signals for MAG and other
acylglycerols the QTOF parameters were optimized. The declustering potential (DP) is a voltage applied
that helps
from clustering,
can introduce
in-source
fragmentation
needstotothe
beorifice
optimized.
Inprevent
these ions
experiments,
the but
DPhigh
wasDP
ramped
fromthe
0 to
200 volts
(V) with a 2 V
[17]. Thus,
the DP value
the ionization
efficiency
and sensitivity,
which
thusTAG
needst17:1
to be were
optimized.
step. The
intensities
of theaffects
molecular
ions of
MAG 17:1,
DAG d17:1,
and
recorded.
In these experiments, the DP was ramped from 0 to 200 volts (V) with a 2 V step. The intensities of the
Figure 3 shows the relatively stable ionization curve of all three acylglycerides with a DP at 60–100 V.
molecular ions of MAG 17:1, DAG d17:1, and TAG t17:1 were recorded. Figure 3 shows the relatively
At DPstable
above
120 V, the
intensity
of MAG
17:1 and
DAG
decreased
while
the intensity
ionization
curve
of all three
acylglycerides
with
a DPd17:1
at 60–100
V. At DPgradually
above 120 V,
the intensity
of TAG
t17:1
continued
to
increase.
Considering
other
impurities
in
the
solutions,
we
thus
selected a
of MAG 17:1 and DAG d17:1 decreased gradually while the intensity of TAG t17:1 continued to increase.
DP of Considering
100 V for our
next
experiments.
other
impurities
in the solutions, we thus selected a DP of 100 V for our next experiments.
3. Effects
of the
declustering potential
on on
ESIESI
ionization
of MAG
DAG
d17:1,
andd17:1,
TAG and
FigureFigure
3. Effects
of the
declustering
potential(DP)
(DP)
ionization
of 17:1,
MAG
17:1,
DAG
t17:1 at a concentration of 1 µM. Spectra were acquired under the following conditions: GS1 = 10, GS2 =
TAG t17:1 at a concentration of 1 µM. Spectra were acquired under the following conditions: GS1 = 10,
10, CUR = 15, Temp = 300 °C.
GS2 = 10, CUR = 15, Temp = 300 ◦ C.
Another parameter that affects ionization efficiency and sensitivity of compounds in ESI-MS
experiments
is ion source
Higher temperatures
helpsensitivity
accelerate the
evaporation process
Another
parameter
thattemperature.
affects ionization
efficiency and
of compounds
in ESI-MS
during
ESI,
but
can
result
in
hydrolysis
of
labile
compounds.
We
performed
experiments
with
a
constant
experiments is ion source temperature. Higher temperatures help accelerate the evaporation process
infusion
of acan
mixture
of MAG
17:1, DAG d17:1,
and compounds.
TAG t17:1 at temperatures
that increased
from 100
during
ESI, but
result
in hydrolysis
of labile
We performed
experiments
with a
to 400 °C. When the temperature was increased from 100 to 300 °C, ionization efficiency, expressed as
constant infusion of a mixture of MAG 17:1, DAG d17:1, and TAG t17:1 at temperatures that increased
peak height of molecular ions, increased linearly (Figure 4). However, peak height leveled off at 400 °C
◦
◦ C, ionization efficiency,
from 100
to 400
When
the
was increased
100 to 300
for DAG
and C.
TAG.
Given
thetemperature
stability of compounds
at lowerfrom
temperatures,
300 °C
was selected as the
expressed
as peak
height offor
molecular
ions, increased linearly (Figure 4). However, peak height leveled
ion source
temperature
further optimization.
off at 400 ◦ C for DAG and TAG. Given the stability of compounds at lower temperatures, 300 ◦ C was
Metabolites 2016, 6, 25
5 of 14
selected as the ion source temperature for further optimization.
Figure 4. Effects of ESI source temperature on ESI ionization of MAG 17:1, DAG d17:1, and TAG t17:1 at
Figure
4. Effects of ESI source temperature on ESI ionization of MAG 17:1, DAG d17:1, and TAG t17:1
a concentration of 1 µM. Spectra were acquired under the following conditions: GS1 = 10, GS2 = 10, CUR
at a concentration of 1 µM. Spectra were acquired under the following conditions: GS1 = 10, GS2 = 10,
= 15, DP = 100 V.
CUR = 15, DP = 100 V.
2.2. Fragmentation Mechanism of MAG, DAG, and TAG
To investigate the fragmentation mechanism and improve product ion sensitivity, MS/MS
fragmentation of MAG 17:1, DAG d17:1, and TAG t17:1 were performed under different collision
energies (CE) ranging from 0 to 100 V. The dominant product ions from each compound were monitored
Metabolites 2016, 6, 25
2.2.
5 of 13
Figure 4. Effects of ESI source temperature on ESI ionization of MAG 17:1, DAG d17:1, and TAG t17:1 at
a concentration of 1 µM. Spectra were acquired under the following conditions: GS1 = 10, GS2 = 10, CUR
Fragmentation Mechanism of MAG, DAG, and TAG
= 15, DP = 100 V.
To investigate the fragmentation mechanism and improve product ion sensitivity, MS/MS
2.2. Fragmentation Mechanism of MAG, DAG, and TAG
fragmentation of MAG 17:1, DAG d17:1, and TAG t17:1 were performed under different collision
To investigate the fragmentation mechanism and improve product ion sensitivity, MS/MS
energies (CE)
ranging from 0 to 100 V. The dominant product ions from each compound were monitored
fragmentation of MAG 17:1, DAG d17:1, and TAG t17:1 were performed under different collision
during energies
infusion.
The optimal CE for the product ion of MAG 17:1 was 15 V (Figure 5A), whereas the
(CE) ranging from 0 to 100 V. The dominant product ions from each compound were monitored
optimalduring
CE forinfusion.
the product
ions of
and
TAGion
was
V and
V,15respectively
(Figures
The optimal
CEDAG
for the
product
of 32
MAG
17:1 35
was
V (Figure 5A),
whereas6A
theand 7A).
optimalion
CE for
the product
ions of
DAG
and 17:1,
TAG was
32 Vd17:1,
and 35 and
V, respectively
(Figures
and acquired
7A).
The product
MS/MS
spectra
for
MAG
DAG
TAG t17:1
were6A
then
at
The product
ion MS/MS
spectra fragmentation
for MAG 17:1, DAG
d17:1, andwere
TAGanalyzed.
t17:1 were then
at spectrum
each
each optimal
CE and
the possible
pathways
Theacquired
MS/MS
of
optimal CE and the possible fragmentation pathways were analyzed. The MS/MS spectrum of MAG 17:1
MAG 17:1 resulted in the dominant product ion at m/z 251.2393 as+ [FA − H2 O + H]+ due to the neutral
resulted in the dominant product ion at m/z 251.2393 as [FA − H2O + H] due to the neutral loss of glycerol
loss of glycerol
(Figure
whereas forDAG
corresponding
andproduct
TAG, the
product ions
(Figure 5B),
whereas5B),
for corresponding
and TAG, theDAG
dominant
ions dominant
[M − FA]+ resulted
+ resulted from the neutral loss of one fatty acid chain (Figures 6B and 7B, respectively).
[M − FA]
from
the neutral loss of one fatty acid chain (Figures 6B and 7B, respectively).
(A) Plot
of intensityof
of fragment
fragment m/z
251.2393
of MAG
with17:1
varying
rolling
collision
energycollision
Figure Figure
5. (A)5.Plot
of intensity
m/z
251.2393
of17:1
MAG
with
varying
rolling
(CE); (B) MS/MS spectra for MAG 17:1 with CE = 15 V; insert: the fragmentation pathway of MAG 17:1
energy (CE); (B) MS/MS spectra for MAG 17:1 with CE = 15 V; insert: the fragmentation pathway
via ESI tandem MS. Spectra were acquired under the following conditions: GS1 = 10, GS2 = 10, CUR = 15,
of MAG
17:1= 300
via°C,
ESIDPtandem
MS.
Spectra
were acquired under the following conditions: GS1 = 10,
Temp
= 100 V, CE
= 15
V.
◦ C, DP = 100 V, CE = 15 V.
GS2
=
10,
CUR
=
15,
Temp
=
300
Metabolites 2016, 6, 25
6 of 14
FigureFigure
6. (A)6. Plot
intensity
m/z
325.2742
ofd17:1
DAG
d17:1
with
varying
rolling
collision
(A) Plot
intensityof
of fragment
fragment m/z
325.2742
of DAG
with
varying
rolling
collision
energy
(B)(B)
MS/MS
spectraspectra
for DAGfor
d17:1
withd17:1
CE = 32
V; insert:
pathway
of DAG d17:1
energy(CE);
(CE);
MS/MS
DAG
with
CE =the32fragmentation
V; insert: the
fragmentation
pathway
viad17:1
ESI tandem
MS.tandem
Spectra were
followingunder
conditions:
= 10, GS2conditions:
= 10, CUR = 15,
of DAG
via ESI
MS.acquired
Spectraunder
werethe
acquired
the GS1
following
GS1 = 10,
Temp = 300 °C, DP = 100 V, CE = 32 V.
GS2 = 10, CUR = 15, Temp = 300 ◦ C, DP = 100 V, CE = 32 V.
Figure 6. (A) Plot intensity of fragment m/z 325.2742 of DAG d17:1 with varying rolling collision energy
(CE); (B) MS/MS spectra for DAG d17:1 with CE = 32 V; insert: the fragmentation pathway of DAG d17:1
via 2016,
ESI tandem
Metabolites
6, 25 MS. Spectra were acquired under the following conditions: GS1 = 10, GS2 = 10, CUR = 15, 6 of 13
Temp = 300 °C, DP = 100 V, CE = 32 V.
Figure7.7. (A) Plot
fragment
m/zm/z
575.5015
of TAG
rolling collision
Figure
Plotintensity
intensityofof
fragment
575.5015
of t17:1
TAGwith
t17:1varying
with varying
rolling energy
collision
(CE);
(B)
MS/MS
spectra
for
TAG
t17:1
with
CE
=
38
V;
insert:
the
fragmentation
pathway
of
TAG
t17:1
energy (CE); (B) MS/MS spectra for TAG t17:1 with CE = 38 V; insert: the fragmentation pathway
via
ESI
tandem
MS.
Spectra
were
acquired
under
the
following
conditions:
GS1
=
10,
GS2
=
10,
CUR
= 15,
of TAG t17:1 via ESI tandem MS. Spectra were acquired under the following conditions: GS1
= 10,
Temp
=
300
°C,
DP
=
100
V,
CE
=
35
V.
◦
GS2 = 10, CUR = 15, Temp = 300 C, DP = 100 V, CE = 35 V.
2.3. Quantification of MAG Using the MS/MSALL
High-resolution analysis of fragment ions using the MS/MSALL strategy was used for accurate
quantification as previously reported [16,18]. To evaluate the linear response of MAG and its dynamic
quantification range based on the MAG-derived fragment ions, we prepared a serial dilution of MAG
17:1 in human plasma and plotted the calibration curves ranging from 2.6 fmol/µL to 40 pmol/µL, with
an R2 regression value of 0.9931 (Figure 8). To assess the reproducibility, we monitored the response of
the synthetic standard, MAG 17:1, with different concentrations in a series of three replicates of human
plasma. The intensity of characteristic fragment m/z 251.2393 resulted in a coefficient of variation (CV)
in the range of 2%–8.5%, except at the lower limit of quantification (LLOQ) concentration where the CV
was 13.2% (Table 1). The low concentration of lipids in the infusion solution is essential for accurate
quantification of lipid molecular species with the same polar head group because at low concentrations
the ionization efficiency of lipids is determined by the polar head group and is independent of fatty
acyl chain length and unsaturation [11,12]. To confirm this for MAG, equal molar MAGs with different
fatty acid chain lengths and numbers of double bonds were profiled based on the neutral loss tandem
MS spectrum at m/z 92.0473 (spectrum was obtained from the acquired MS/MSALL data). All MAG
species with different chain length and double bonds showed nearly identical ionization efficiency
(Figure 9A) and the ratio of the MAG species to MAG 17:1 was around 1 (Figure 9B). For biological
samples, selected concentrations of the internal standard, MAG 17:1, were spiked into plasma or
adipose tissue homogenates before extraction. Sample preparation was performed as described in the
materials and methods, and the samples were diluted in the running buffer to ensure the dynamic
range and ionization efficiency. Another prerequisite for accurate quantification for MS/MSALL or
any other data independent acquisition (DIA) is the database searching and accurate quantitation
based on optimal ion/adduct selection. An in-house database for MAGs was constructed based on the
information of MAG high resolution m/z of molecular ions and diagnostic fragments (Table 2).
homogenates before extraction. Sample preparation was performed as described in the materials and
methods, and the samples were diluted in the running buffer to ensure the dynamic range and ionization
efficiency. Another prerequisite for accurate quantification for MS/MSALL or any other data independent
acquisition (DIA) is the database searching and accurate quantitation based on optimal ion/adduct
selection. An in-house database for MAGs was constructed based on the information of MAG high
Metabolites 2016, 6, 25
7 of 13
resolution m/z of molecular ions and diagnostic fragments (Table 2).
Metabolites 2016, 6, 25
8 of 14
Table 1. Reproducibility of the MS/MSALL technique for MAG.
Concentration
Sample A
1,360,500
397,250
105,950
40 pmol/µL
8 pmol/µL
1.6 pmol/µL
Peak Intensity
Sample B
Sample C
1,454,000
1,568,500
421,500
356,450
105,590
94,170
Mean (n = 3)
CV (%)
1,461,000
391,733
101,903
7.13
8.39
6.57
Figure
plot
response
of of
MAG
vs.vs.
thethe
concentration
of of
infused
MAG
17:1
yielded
a
Figure8.8.The
The
plotofofESI-MS/MS
ESI-MS/MS
response
MAG
concentration
infused
MAG
17:1
yielded
0.3 pmol/µL
33,190
32,400
36,615
34,068
6.58
2 = 20.9993) when spiked in human plasma. The insert table shows the related quantitative
linear
line
(R
a linear line (R = 0.9993) when spiked in human plasma. The insert table shows the related quantitative
64 fmol/µLthe lower limit
9840
9464.5
9841.5
9715 MS/MS data
2.24
parameters.
parameters.LLOQ,
LLOQ, the lower limitofofquantification.
quantification.The
Thefull
fullhigh-resolution
high-resolution MS
MS and MS/MS
data were
were
13using
fmol/µL
4562.5
5110
4837
5.66
ALL ALL
mode
under
the 4839
following
conditions:
GS1 =GS1
10, GS2
10, CUR
15, Temp
collected
thethe
MS/MS
collected
using
MS/MS
mode
under
the following
conditions:
= 10,= GS2
= 10,=CUR
= 15,
2.6
fmol/µL
2323
2807
3020
2716
13.2
= Temp
300 °C,
=◦100
V, CE
= 15
± 10
V. V ± 10 V.
=DP
300
C, DP
= 100
V,VCE
= 15
(A)
(B)
100
353.2
355.2
331.2
0
300
320
340
MAG 18:3
MAG 18:2
MAG 18:0
359.2
MAG 17:1
50
MAG 16:0
Relative Intensity (%)
343.2
360
380
400
Precursor, Da
Figure 9. (A) Neutral loss (m/z 92.0473) tandem mass spectra of an equimolar mixture (10 fmol/µL);
Figure 9. (A) Neutral loss (m/z 92.0473) tandem mass spectra of an equimolar mixture (10 fmol/µL); (B)
(B) The
of individual
MAG
species
to MAG
3). The
high-resolution
The
peakpeak
areaarea
ratioratio
of individual
MAG
species
to MAG
17:117:1
(n =(n
3).= The
full full
high-resolution
MS MS
and
ALL mode under the following conditions: GS1 = 10,
and
MS/MS
data
were
collected
using
the
MS/MS
ALL
MS/MS data were collected using the MS/MS mode under the following conditions: GS1 = 10, GS2 = 10,
◦
GS2 == 15,
10, Temp
CUR == 15,
= =300
100± V,
= 15neutral
± 10 V.loss
Thescan
neutral
lossspectra
scan MS/MS
spectra
CUR
300Temp
°C, DP
100 C,
V, DP
CE == 15
10 CE
V. The
MS/MS
was generated
was
generated
from
the
collected
data
retrospectively.
from the collected data retrospectively.
ALL technique for MAG.
Table 1. Reproducibility
of theSpecies
MS/MSDatabase.
Table 2. MAG
MAG species
Concentration
Sample A
MAG 14:1
40 pmol/µL
1,360,500
8 pmol/µLMAG 14:0397,250
MAG 16:1105,950
1.6 pmol/µL
0.3 pmol/µL
MAG 16:033,190
64 fmol/µL
9840
IS MAG 17:1
13 fmol/µL
4562.5
MAG 18:3 2323
2.6 fmol/µL
MAG 18:2
MAG 18:1
MAG 18:0
MAG 20:4
MAG 20:3
Peak[M
Intensity
+ H]+
Sample B
301.3
1,454,000
303.3
421,500
329.3
105,590
32,400
331.3
9464.5
343.3
4839
353.3
2807
355.3
357.3
359.3
379.3
381.3
Diagnostic Fragment
3)+
CV (%)
[FA Mean
+ H − (n
H2=O]
Sample C
209.1906
1,568,500
1,461,000
7.13
211.2062
356,450
391,733
8.39
237.2219
94,170
101,903
6.57
36,615
34,068
6.58
239.2376
9841.5
5110
3020
9715
251.2369
4837
261.2219
2716
263.2376
265.2532
267.2689
287.2376
289.2532
2.24
5.66
13.2
Metabolites 2016, 6, 25
8 of 13
Table 2. MAG Species Database.
MAG Species
MAG 14:1
MAG 14:0
Metabolites 2016,MAG
6, 25 16:1
MAG 16:0
IS MAG 17:1
MAG 18:3
MAG 18:2
MAG
MAG
18:1species
MAG 18:0
MAG 20:2
MAG 20:4
MAGMAG
20:3 20:1
MAGMAG
20:2 20:0
MAG 20:1
MAG 22:6
MAG 20:0
MAGMAG
22:6 22:5
MAGMAG
22:5 22:4
MAGMAG
22:4 22:3
MAG 22:3
MAGMAG
22:2 22:2
MAGMAG
22:1 22:1
MAGMAG
22:0 22:0
[M + H]+
Diagnostic Fragment [FA + H − H2 O]+
301.3
303.3
329.3
331.3
343.3 Table 2. Cont.
353.3
355.3
+
357.3[M + H]
359.3
383.3
379.3
381.3 385.3
383.3 387.4
385.3
403.3
387.4
403.3 405.3
405.3 407.3
407.3 409.3
409.3
411.4 411.4
413.4 413.4
415.4 415.4
209.1906
211.2062
237.2219
239.2376
251.2369
261.2219
263.2376Fragment
Diagnostic
265.2532
[FA
+ H − H2O]+
267.2689
291.2689
287.2376
293.2845
289.2532
291.2689
295.3002
293.2845
311.2376
295.3002
313.2532
311.2376
313.2532
315.2689
315.2689
317.2845
317.2845
319.3002
319.3002
321.3158
321.3158
323.3315
323.3315
9 of 14
ALL method was applied to analyze the profile of MAGs in three representative
TheDIA
DIAMS/MS
MS/MSALL
The
method was applied to analyze the profile of MAGs in three representative
sample types
types that
bebe
routinely
used
or commonly
investigated
for lipid
Human
sample
thatwould
wouldeither
either
routinely
used
or commonly
investigated
formetabolism.
lipid metabolism.
ALL
plasma
and
mouse
brown
and
white
adipose
tissues
were
selected
for
analysis
using
the
MS/MS
Human plasma and mouse brown and white adipose tissues were selected for analysis using the
method.ALL
Themethod.
dominant
MAG
species MAG
in human
plasma
were saturated
18:0 and MAG
MS/MS
The
dominant
species
in human
plasma MAG
were saturated
MAG16:0,
18:0others
and
consisted
of
medium-chain
fatty
acids.
Compared
to
mouse
white
adipose
tissue,
brown
adipose
tissue
MAG 16:0, others consisted of medium-chain fatty acids. Compared to mouse white adipose tissue,
showed
a distinct
pattern
of MAG
withpattern
notablyofmore
polyunsaturated
fattypolyunsaturated
acid MAGs, such
as MAG
brown
adipose
tissue
showed
a distinct
MAG
with notably more
fatty
acid
22:3 and MAG 22:6 (Figure 10).
MAGs, such as MAG 22:3 and MAG 22:6 (Figure 10).
Figure
Profileofof
MAG
molecular
species
obtained
from
(A) human
plasma
and (B)
mouse
Figure 10. Profile
MAG
molecular
species
obtained
from (A)
human
plasma and
(B) mouse
brown
and
brown
and white
adipose
tissue
The means
data represent
± and
S.E.6ofreplicates
n = 6 mice
and
white adipose
tissue
samples.
The samples.
data represent
± S.E. of nmeans
= 6 mice
of human
ALLcollected
6plasma.
replicates
human
plasma. The
full MS/MS
high-resolution
and MS/MS
were
using
Theof
full
high-resolution
MS and
data wereMS
collected
using thedata
MS/MS
mode under
the
ALL
◦ C,
following
conditions:
= 10,
GS2
= 10, CUR
= 15, TempGS1
= 300
°C, GS2
DP ==100
CE ==15
± 10
V. The
neutral
the
MS/MS
modeGS1
under
the
following
conditions:
= 10,
10,V,CUR
15,
Temp
= 300
loss=scan
MS/MS
generated
collected
data
retrospectively.
DP
100 V,
CE = spectra
15 ± 10was
V. The
neutralfrom
lossthe
scan
MS/MS
spectra
was generated from the collected
data retrospectively.
3. Discussion
MAGs are found in trace amounts in blood and tissues and play an essential role in metabolic
homeostasis and diverse physiological functions [3,4,7]. Mass spectrometry based platforms have
advanced significantly in the past few years, providing opportunities to develop robust high throughput
analysis of MAGs in diverse sample types with improved sensitivity and identification. Herein, we have
adapted and optimized a DIA MS/MSALL shotgun lipidomics method for the analysis of MAGs that can
Metabolites 2016, 6, 25
9 of 13
3. Discussion
MAGs are found in trace amounts in blood and tissues and play an essential role in metabolic
homeostasis and diverse physiological functions [3,4,7]. Mass spectrometry based platforms have
advanced significantly in the past few years, providing opportunities to develop robust high
throughput analysis of MAGs in diverse sample types with improved sensitivity and identification.
Herein, we have adapted and optimized a DIA MS/MSALL shotgun lipidomics method for the analysis
of MAGs that can be utilized for various biological samples and for broader use in defining the role of
metabolic intermediates in regulating physiological function.
Although MAG, DAG, and TAG all belong to the acylglycerol family, which are the most abundant
neutral lipids in mammals, their ESI-MS behavioral properties are quite different. MAG with only
one fatty acid chain preferentially ionizes to form [M + H]+ as the predominant molecular ion even
in the presence of ammonium acetate, whereas DAG and TAG with two and three fatty acid chains,
respectively, easily form ammonium ion adducts. Also, the ratio of the intensity of molecular ions
is roughly proportional to the number of fatty acid chains (1:2:3). The different ESI-MS behavior
of acylglycerols demonstrates that the molecular structure of the acylglycerols can influence its ESI
behavior. Furthermore, the hydrophobic portion of acylglycerol can determine the ESI response
because analytes with greater hydrophobicity have higher affinities for the surface of ESI droplets,
which in turn produces higher ESI signals [19]. In addition, the different molecular structure of
acylglycerols and the composition of the ESI solution influences the MS/MS fragmentation pathways
and mechanism [9,11,12,20]. Alkali metal adduct ions of acylgylcerols usually produce the specific
fragments due to the neutral loss of fatty acids [9,12]. Our results are consistent with DAG and TAG
ammonium adduct molecular ions, whereas MAG displayed a different fragmentation pattern due to
the neutral loss of glycerol.
Shotgun lipidomics exploits the chemical and physical properties of lipids to facilitate the
high throughput global MS-based analysis of a lipidome directly from organic extracts of biological
samples [12,21]. The lipidome is profiled based on the specific head groups and fatty acid chains
either through precursor ion scans, neutral loss scans, or multiple reaction monitoring experiments,
primarily carried out on triple quadrupole (QQQ) instruments [12,22,23]. However, the disadvantage
of QQQ analyses is that they require the pre-selection of characteristic ions and are highly targeted
due to the vast number of theoretical ions that have to be pre-selected. Instead, data independent
acquisition methods, such as the MS/MSALL technique, have proven successful for untargeted lipid
analysis [13,16,18]. A key point in MS/MSALL analysis is selection of the optimal collision energy of
molecular ions to produce the most abundant distinct fragment ions in each window. For accurate
quantification of MAGs and other lipids using the MS/MSALL strategy, similar to all other the shotgun
lipidomics methods [11,24,25], it is favorable to quantitate lipids when at a low concentrations in
solution as at low concentrations lipid-lipid interactions are negligible and ionization efficiency of
the molecular species of the same lipid class is predominantly determined by the nature of the polar
head group. For MAG, we illustrated at a low concentration of 10 fmol/µL different MAG species of
various chain lengths and number of double bonds had similar peak intensities and areas under the
optimal ESI/MS conditions. The linear range for quantification of MAGs was between the fmol/µL
and pmol/µL range. The low concentration range requires the appropriate dilution of the lipid extracts
for accurate quantification [11,12,16]. One limitation of the MS/MSALL method for MAG analysis is
that it does not differentiate the regio-isomers, i.e. the sn-position of the fatty acid chain in the glycerol
backbone. This can be resolved by using gas chromatography, liquid chromatography, or ion mobility
coupled to mass spectrometry [7,10,26].
The MS/MSALL method for MAG was successfully applied as demonstrated by the results from
MAG profiling of human plasma and mouse white and brown adipose tissues. Saturated fatty acid
MAG species, such as MAG 18:0 and MAG 16:0 dominate MAG components in all three samples. It is
notable that white and brown adipose tissue showed differences in their pattern of MAG species, which
may explain functional differences between them. For example, white and brown adipose tissues differ
Metabolites 2016, 6, 25
10 of 13
significantly based on their structure and physiology [27,28]. Specifically, the primary purpose of white
adipose tissue is to store and release energy in the form of free fatty acids, while the essential function
executed by brown adipose tissue is the expenditure of fatty acid-derived energy for maintenance of
the organism’s thermal stability [29–31]. Our MAG analysis showed a similar distribution for a large
amount of fatty acids, such as MAG 16:0, MAG 18:0, MAG 18:1, and MAG 18:2, between white and
brown adipose tissue. These MAG species can be immediately accessed by enzymes and provide the
lipid source for either energy needs or thermal balance [32]. Interestingly, in brown adipose tissue,
there were certain long-chain polyunsaturated fatty acids, such as MAG 22:6 and MAG 22:3, which
may be released by monoacyglycerol lipase and converted into bioactive lipid mediators.
In summary, we established an MS/MSALL method for accurate identification and quantification
of MAG species from complex biological samples through the optimization of ESI/MS parameters.
Compared with other acylglycerols, DAG and TAG, MAG demonstrated distinct MS and tandem MS
ionization conditions, which were exploited for MAG identification and quantification in the biological
samples. The method developed here can be applied to broader studies for measurement of MAG in
biofluids and diverse sample types in a robust and high throughput approach.
4. Experimental Procedures
4.1. Materials
MAG 17:1, MAG 16:0, MAG 18:0, MAG 18:2, MAG 18:3, DAG d17:1, and TAG t17:1 were
purchased from Nu-Chek Prep Inc (Waterville, MN, USA). All solvents were of HPLC or LC/MS grade
and were purchased from Fisher Scientific (Waltham MA, USA) or VWR International (Radnor, PA,
USA). Human plasma was purchased from Bioreclammation (Baltimore, MD, USA) and mouse brown
and white adipose tissues were obtained from Joslin Diabetes Center. All animal procedures were
approved by the Institutional Animal Use and Care Committee at Joslin Diabetes Center.
The stock solutions of all acylglycerols were prepared using chloroform:methanol (1:1, v/v)
and the working solutions were prepared by diluting the stock solutions using the running buffer
(see below).
4.2. Sample Preparation and Extraction
Human plasma samples were thawed on ice. Before extraction, an appropriate amount (1.65 nmol)
of the internal standard, MAG 17:1 was added into 25 µL of plasma. Then, 4 mL chloroform:methanol
(1:1, v/v) and 2 mL LiCl solution (50 mmol) were added to each sample and the lipid extraction was
performed as previously described [16,33]. Specifically, the extraction homogenate was vortexed and
centrifuged at 2500 x g for 5 min. The chloroform layer of each extract mixture was carefully removed
and collected. An additional 2 mL chloroform was added into the MeOH/aqueous layer of each test
tube. After centrifugation, the chloroform layer from each individual sample was combined and dried
under a nitrogen stream. Each individual residue was then re-suspended in 4 mL chloroform/methanol
(1:1), back-extracted against 2 mL LiCl aqueous solution (10 mmol), and the extract was dried as
described above. Such lipid extraction was automated using a customized sequence on a Hamilton
Robobtics STARlet system (Reno, NV, USA) to meet the high throughput requirements. Finally,
lipid extracts were dried under nitrogen and reconstituted in 68 µL chloroform:methanol (1:1, v/v).
Samples were flushed with nitrogen and stored at −20 ◦ C.
To investigate the dynamic quantification range and assay reproducibility, the synthetic standard,
MAG 17:1, was spiked at different serial concentrations into 25 µL human plasma prior to extraction
and experiments were performed in triplicates.
For tissue analysis, 10–20 mg of mouse adipose tissue was transferred to an OMNI bead tube
with 0.8 mL 10× diluted PBS and was homogenized for 1.5 min at 4 ◦ C using the OMNI Cryo Bead
Ruptor 24 (OMNI international, Inc., Kennesaw, GA, USA). An aliquot of the homogenate was taken
for protein analysis using a BCA assay and 1 mg of sample based on protein concentration was added
Metabolites 2016, 6, 25
11 of 13
to internal standards and 4 mL chloroform:methanol (1:1, v/v) for robotic extraction [34]. Lipid extracts
were dried under nitrogen and reconstituted in 400 µL chloroform:methanol (1:1, v/v). Samples were
flushed with nitrogen and stored at −20 ◦ C.
4.3. Direct Infusion Quadruple Time of Flight (QTOF) Mass Spectrometry
The concentrated samples (both plasma and tissue) were diluted 50× in isopropanol:
methanol:acetonitrile:H2 O (3:3:3:1, by vol.) with 2 mM ammonium acetate in LC vials, and 50 µL
diluted lipid extract was automatically loaded and directly delivered to the ESI source using an Ekspert
microLC 200 system (SCIEX, Framingham, MA, USA) with a flow rate of 6 µL/min on a customized
sample loop. The MS/MSALL acquisition experiment was performed on a SCIEX TripleTOF 5600+
(SCIEX, Framingham, MA, USA). ESI source parameters included nebulizing gases (GS) GS1 at 10,
GS2 at 10, curtain gas (CUR) at 15, positive mode ion spray voltage at 5000, declustering potential
at 100 V, and temperature 300 ◦ C. The atmospheric-pressure chemical ionization (APCI) probe and
inlet were connected to a calibrant pump which delivers mass calibration solution for MS and MS/MS.
The MS/MSALL data acquisition was controlled with Analyst® TF 1.5.1 software (SCIEX). An MS
range was 200–1200 m/z at an accumulation time of 300 ms, followed by 1000 product ion scans with
1000 precursors evenly spaced from m/z 200.051 to m/z 1200.051, measuring across m/z 100–1500 with
the accumulation time 300 ms each. Total time for one MS/MSALL acquisition was 5.5 min. The optimal
collision energy (CE) for MAG MS/MS fragmentation was 15 ± 10 eV.
Acknowledgments: This work was supported in part by R01DK077097 and R01DK102898 (to Y.-H.T.), and
P30DK036836 (to Joslin Diabetes Center’s Diabetes Research Center, DRC) from the National Institute of Diabetes
and Digestive and Kidney Diseases. M.D.L was supported by NIH fellowships (T32DK007260 and F32DK102320).
We thank Khampaseuth Thapa for editing the manuscript.
Author Contributions: F.G. and M.A.K. designed and directed the research and co-wrote the paper. F.G., J.M.,
E.Y.C., H.R., R.S., and N.R.N. carried out experiments, analyzed data, and co-wrote the paper. M.D.L and Y.-H.T.
provided the mouse adipose tissue samples and helped interpret the results.
Conflicts of Interest: This study was funded by Berg, LLC. F.G., J.M., E.Y.C., H.R., R.J., N.R.N., and M.A.K. are
current employees of Berg, LLC and have stock options.
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