Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: Discovery of acyl-dephospho-CoAs

Article abstract of DOI:10.1194/jlr.D045112

A metabolomic approach to selectively profile all acyl-CoAs was developed using a programmed multiple reaction monitoring (MRM) method in LC-MS/MS and was employed in the analysis of various rat organs. The programmed MRM method possessed 300 mass ion transitions with the mass difference of 507 between precursor ion (Q1) and product ion (Q3), and the precursor ion started from m/z 768 and progressively increased one mass unit at each step. Acyl-dephospho-CoAs resulting from the dephosphorylation of acyl-CoAs were identified by accurate MS and fragmentation. Acyl-dephospho-CoAs were also quantitatively scanned by the MRM method with the mass difference of 427 between Q1 and Q3 mass ions. Acyl-CoAs and dephospho- CoAs were assayed with limits of detection ranging from 2 to 133 nM. The accuracy of the method was demonstrated by assaying a range of concentrations of spiked acyl-CoAs with the results of 80-114%. The distribution of acyl-CoAs reflects the metabolic status of each organ. The physiological role of dephosphorylation of acyl-CoAs remains to be further characterized. The methodology described herein provides a novel strategy in metabolomic studies to quantitatively and qualitatively profile all potential acyl-CoAs and acyl-dephospho-CoAs. Copyright

Full text of DOI:10.1194/jlr.D045112

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methods
Novel approach in LC-MS/MS using MRM to
generate a full profile of acyl-CoAs: discovery of
acyl-dephospho-CoAs
Qingling Li,* Shenghui Zhang,* Jessica M. Berthiaume,* Brigitte Simons,†
and Guo-Fang Zhang1,*
Department of Nutrition,* Case Western Reserve University, Cleveland, OH 44106; and AB SCIEX,†
Concord, Ontario L4K 4V8, Canada
Abstract A metabolomic approach to selectively profile all
acyl-CoAs was developed using a programmed multiple re-
action monitoring (MRM) method in LC-MS/MS and was
employed in the analysis of various rat organs. The pro-
grammed MRM method possessed 300 mass ion transitions
with the mass difference of 507 between precursor ion (Q1)
and product ion (Q3), and the precursor ion started from
m/z 768 and progressively increased one mass unit at each
step. Acyl-dephospho-CoAs resulting from the dephospho-
rylation of acyl-CoAs were identified by accurate MS and
fragmentation. Acyl-dephospho-CoAs were also quantita-
tively scanned by the MRM method with the mass difference
of 427 between Q1 and Q3 mass ions. Acyl-CoAs and de-
phospho-CoAs were assayed with limits of detection ranging
from 2 to 133 nM. The accuracy of the method was dem-
onstrated by assaying a range of concentrations of spiked
acyl-CoAs with the results of 80–114%. The distribution of
acyl-CoAs reflects the metabolic status of each organ. The
physiological role of dephosphorylation of acyl-CoAs re-
mains to be further characterized. The methodology de-
scribed herein provides a novel strategy in metabolomic
studies to quantitatively and qualitatively profile all poten-
tial acyl-CoAs and acyl-dephospho-CoAs.—Li, Q., S. Zhang,
J. M. Berthiaume, B. Simons, and G-F. Zhang. Novel ap-
proach in LC-MS/MS using MRM to generate a full profile
of acyl-CoAs: discovery of acyl-dephospho-CoAs. J. Lipid
Res. 2014. 55: 592–602.
Acyl-CoAs are a class of important molecules that play
essential roles in many physiological processes (1), such as
fatty acid oxidation, lipid synthesis/remodeling, ketone
body synthesis, xenobiotic metabolism, and signaling path-
ways. Classically, acyl-CoAs such as free CoA, acetyl-CoA,
and malonyl-CoA are recognized as regulators of meta-
bolic flux. The ratio of acetyl-CoA versus free CoA tightly
regulates glycolysis and fatty acid oxidation. Malonyl-CoA
attenuates fatty acid oxidation by inhibiting acyl-CoA trans-
port into the mitochondrion for oxidation and is utilized
in fatty acid synthesis when its concentration is elevated
(2). Many proteins and genes are dynamically regulated
by deacylation and acylation via various acyl-CoAs, such
as acetyl-CoA, succinyl-CoA, palmitoyl-CoA, etc. (3). How-
ever, acyl-CoAs present in the cell are diverse and may in-
volve molecules beyond fatty acids and their oxidized
derivatives. The metabolism of xenobiotics can also lead to
the formation of acyl-CoAs, as demonstrated in our previ-
ous work on the metabolism of 4-hydroxy acids from C4 to
C
11 in perfused rat liver. The identification of these novel
acyl-CoAs extends our understanding of the new catabolic
pathways involved in the disposal of 4-hydroxy acids in-
cluding drugs of abuse and lipid peroxidation products
(4–8).
Acyl-CoAs are exclusive to the intracellular metabolites,
and the profile of these biomolecules is indicative of the lo-
cal metabolic status. Each organ has its specific physiologi-
cal roles with different energetic demands, and therefore,
would be expected to exhibit a unique acyl-CoA profile.
The heart preferentially utilizes fatty acids to meet the ATP
demand of mechanical contraction. Glucose and/or ketone
bodies serve as the primary substrate of the brain for energy
Supplementary key words multiple reaction monitoring • liquid
chromatography • mass spectrometry • rat organs
This publication was made possible by the Case Western Reserve University/
Cleveland Clinic CTSA (Clinical & Translational Science Awards) Grant UL1
RR024989 from the National Center for Research Resources to G-F.Z. and a
component of the National Institutes of Health and National Institutes of Health
Roadmap for Medical Research and AHA (American Heart Association) Award
12GRNT12050453 to G-F.Z. This work was supported, in whole or in part,
by National Institutes of Health Roadmap Grants R33DK070291 and
R01ES013925 (to H. Brunengraber, Nutrition Department, Case Western Re-
serve University). This work was also supported by a grant from the Cleveland
Mt. Sinai Health Care Foundation.
Abbreviations: CE, collision energy; DP, declustering potential;
LOD, limit of detection; LOQ, limit of quantitation; MRM, multiple
reaction monitoring; RSD, relative standard deviation.
1 To whom correspondence should be addressed.
Manuscript received 23 October 2013 and in revised form 10 December 2013.
e-mail: gxz35@case.edu
Published, JLR Papers in Press, December 20, 2013
DOI 10.1194/jlr.D045112
The online version of this article (available at http://www.jlr.org)
contains supplementary data in the form of six figures and one table.
Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.
592
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propionyl-CoA and [2,2,3,3,4,4,5,5,5-2H9]pentanoyl-CoA were syn-
thesized according to our previous work (4–6, 8). The aqueous
stock solutions of acyl-CoAs were stored at Ϫ80°C.
metabolism. Both fatty acid oxidation and synthesis are
highly active in the liver. The profile or fingerprint of the
acyl-CoAs across these tissues should broadly reflect the
metabolic preferences of these organs, but may also reveal
additional roles of acyl-CoAs in cellular processes.
Animal experiments
Adult male Sprague-Dawley rats (300–350 g) were fed ad libi-
tum for 8–12 days with standard laboratory chow prior to the ex-
periments. All experiments were performed in accordance with
the Institutional Animal Care and Use Committee at Case West-
ern Reserve University.
Fed rats were anesthetized with 5% isoflurane in an induction
chamber, and maintained via facial mask at 2% during the surgi-
cal procedure. A median laparotomy was performed and a bolus
of heparin (500 U/kg) was given via the inferior vena cava. Liv-
ers, hearts, kidneys, and brains were quickly dissected and frozen
in liquid nitrogen until further analysis.
LC-MS/MS is the most sensitive method to analyze acyl-
CoAs and various LC-MS or LC-MS/MS approaches have
been reported [for a full review see (1)]. However, most
methods only assay either short-, medium-, or long-chain
acyl-CoAs (9–19). The best approach for acyl-CoA quanti-
tation, so far, is to measure the target acyl-CoAs from short
to long carbon chain length in a single method (20, 21).
However, no methodology previously described allows
quantitative profiling of acyl-CoAs for the purpose of tar-
geted and untargeted acyl-CoA analysis. The challenges of
profiling acyl-CoAs are as follows: i) Acyl-CoAs are a large
category of metabolites that are present at a range of con-
centrations. ii) Acyl-CoAs vary widely in the polarity of the
acyl moiety challenging LC method development. Alka-
line mobile phases are mostly employed to improve peak
shapes of long-chain acyl-CoAs (20, 22), however, high pH
is deleterious for silica-based C18 columns and greatly di-
minishes the lifespan. iii) Not all acyl-CoAs have authentic
compounds. iv) Multiple reaction mornitoring (MRM) or
selected reaction monitoring is a sensitive MS/MS ap-
proach for targeted analysis, but limits broad profiling.
Although challenges of profiling acyl-CoAs exist, com-
mon fragmentation patterns of acyl-CoAs in LC-MS/MS
were found by us and others (4, 6, 23, 24), and these com-
mon fragmentation patterns enable broad profiling. In this
work, a novel MRM method was developed to quantitatively
scan acyl-CoAs in different rat organs according to the char-
acteristic fragmentation pattern of acyl-CoAs in MS/MS.
Dephospho-CoA and ATP are substrates of the last step
of CoA synthesis catalyzed by dephospho-CoA kinase (25).
Several acyl-dephospho-CoAs have been synthesized in
vitro (26, 27), and valproyl-dephospho-CoA has been iden-
tified in isolated rat liver mitochondria from CoA trapping
reagent (valproate), suggesting that both the phospho-
and dephospho-CoA forms are biologically active (28). The
absence of the 3′-phosphate group on the ribose moiety of
acyl-dephospho-CoAs is the only difference compared with
their corresponding acyl-CoAs. A similar MRM approach
was also developed to quantitatively scan acyl-dephospho-
CoAs in the rat organs.
Preparations of acetyl-dephospho-CoA and
butyryl-dephospho-CoA
To demonstrate that dephospho-CoA is a substrate of acetyl-
CoA synthetase and to prepare authentic acyl-dephospho-CoA,
the synthesis of acetyl-dephospho-CoA and butyryl-dephospho-
CoA by acetyl-CoA synthetase was assayed (29). In microcentri-
fuge tubes, 10 mM sodium acetate or sodium butyrate, 10 mM
MgCl2, 50 mM KH2PO4, 10 mM ATP, and 0.7 mM dephospho-
CoA were mixed into a final volume of 100 ␮l (pH 7.4). Acetyl-
CoA synthetase (0.02 U) was added to the mixture to start the
reaction at 37°C. The reaction was quenched by 500 ␮l ace-
tonitrile after 1 h and enzyme was removed by centrifugation at
800 g for 15 min. The supernatant was dried and 100 ␮l Milli-Q
water was added to redissolve the residue for LC-MS/MS
analysis.
Sample preparation
Acyl-CoAs and acyl-dephospho-CoAs in various tissues were ex-
tracted using our previous methods (4–6, 8). Powdered tissues
(250 mg), spiked with mixtures of internal standard {[2,2,3,3,3-
2H5]propionyl-CoA (0.2 nmol), [2,2,3,3,4,4,5,5,5-2H9]pentanoyl-
CoA (0.2 nmol), and heptadecanoyl-CoA (2 nmol)}, were
homogenized for 1 min using a Polytron homogenizer after add-
ing 4 ml of extraction buffer (methanol/water 1:1 containing 5%
acetic acid). The supernatant was further purified by a 3 ml ion
exchange cartridge packed with 300 mg of 2-(2-pyridyl)ethyl sil-
ica gel (Sigma). The cartridge was preactivated with 3 ml metha-
nol, and then equilibrated with 3 ml of extraction buffer. Another
3 ml of extraction buffer was loaded to clean the sample after
sample loading. The acyl-CoAs trapped on the silica gel cartridge
were eluted by i) 3 ml of a 1:1 mixture of 50 mM ammonium
formate (pH 6.3) and methanol, ii) 3 ml of a 1:3 mixture of
50 mM ammonium formate (pH 6.3) and methanol, and iii) 3 ml
methanol. The combined effluent was dried with N2 gas and
stored at Ϫ80°C until LC-MS/MS analysis. The dried sample was
used for all assays by TripleTOF 5600+ and 4000 QTRAP mass
spectrometers.
The purpose of this work was to develop a MRM method to
scan and quantitate acyl-CoAs and possibly acyl-dephospho-
CoAs. With the developed method, we can quantitatively
map all acyl-CoAs for both targeted and untargeted me-
tabolomic studies.
Acyl-dephospho-CoA identified by TripleTOF 5600+
Dried samples were dissolved in 100 ␮l Milli-Q water and cen-
trifuged for 15 min at 800 g to remove undissolved material. Two
microliters of sample were injected into a 3C18-EP-120 column
(0.5 × 50 mm, 3.0 ␮m, 120 Å) with the column oven temperature
at 40°C. The LC-MS/MS system was a microLC (Ekspert™ mi-
croLC 200 system, AB SCIEX, Concord, ON, Canada) with a
TripleTOF 5600+ system (AB SCIEX). The temperature of the
autosampler was set at 5°C and the flow rate was 40 ␮l/min. A
gradient elution method was adopted as follows: 100% mobile
METHODS
Materials
Acyl-CoAs from short to long carbon chain length (free CoA
to C20), dephospho-CoA, and heptadecanoyl-CoA (internal stan-
dard) were purchased from Sigma (St. Lois, MO). [2,2,3,3,3-2H5]
Acyl-(dephospho)-CoA profile by LC-MS/MS
593

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phase A (25 mM ammonium formate in 98% water and 2% ace-
into diluted rat heart homogenates (n = 4–5) and evaluating by
LC-MS/MS analysis. The diluted rat heart homogenates were
prepared by continuous dilution with extraction buffer until no
acyl-CoAs were detected.
tonitrile, pH 8.2) was maintained from 0 to 0.3 min, mobile phase
B (98% acetonitrile and 2% water, 5 mM ammonium formate)
was increased linearly to 30% from 0.3 to 2.2 min and increased
to 55% from 2.2 to 12 min, and finally increased to 100% within
1 min. The 100% mobile phase B was maintained for 4 min to
clean the column, and then was returned to the initial condition
in 0.5 min followed by column equilibration for 3 min. The total
run time was 20.5 min. The TripleTOF 5600+ mass spectrometer
was set to electrospray ionization mode with an ion spray voltage
of 5.5 kV, curtain gas of 25 psi, gas 1 of 60 psi, gas 2 of 30 psi, and
an interface heater temperature of 350°C. The m/z of the TOF
mass scan was from 300 to 1,250 with an accumulation time of 0.6 s,
a declustering potential (DP) of 60 V, and a minimum collision
energy (CE) voltage of 5 V. The data-independent workflow
mode (SWATH acquisition) was set up to acquire full scan MS/
MS data of precursor ions (m/z from 100 to 1,250) in 60 amu in-
crements. The DP and CE for SWATH acquisition were 60 and
50 V, respectively.
Statistical analyses
The heat map of average concentrations of acyl-CoAs and acyl-
dephospho-CoAs was processed by PermutMatrix (Montpellier
Laboratory, France) (30). The significant P value for the correla-
tion is set at <0.05. Statistical differences were tested using a
paired t-test (Graph Pad Prism software, version 3).
RESULTS
All acyl-CoAs were determined to have the following
common fragmentation patterns in positive electrospray
ionization-MS/MS (Fig. 1): i) fragment m/z 428 that rep-
resents the CoA moiety, and ii) a neutral loss of 507 which
is the 3′-phosphonucleoside diphosphate fragment. The
fragment of acyl-CoAs from the neutral loss of 507 was
the most abundant fragment, and this fragment was used
for acyl-CoA MRM assays. DP and CE are essential param-
eters in MS/MS and are very similar for all acyl-CoAs
from short- to long-chain length. Hence, a programmed
MRM method was designed to scan all acyl-CoAs. The
precursor ion in Q1 started from 768 (free CoA, the
smallest acyl-CoAs), and progressively increased to 1,100
(m/z from 770 to 807 was skipped in this work because
there are no known acyl-CoAs from animal tissues located
in this mass range, but it can be added if it is needed) by
one mass unit increase at each step. The corresponding
product ion in Q3 was the Q1 minus 507 and started from
261 (free CoA) to 593. About 300 ion transitions were
determined to sufficiently cover the range of known acyl-
CoAs, but can be extended to a higher mass range using
a second method.
In addition to classically recognized acyl-CoAs, a subset
of acyl-CoA analogs was identified in TripleTOF 5600+ that
attracted our attention and warranted further investiga-
tion. The metabolites of interest had the following char-
acteristics: i) a fragment from the neutral loss of 427
(acyl-CoAs have the similar fragment, but from a neutral
loss of 507); ii) a common fragment at m/z 348 (acyl-CoAs
have a common fragment at m/z 428, 80 amu higher than
m/z 348); iii) precursor ions of metabolites with 80 amu
less than the corresponding acyl-CoAs; and iv) a longer
retention time compared with the corresponding acyl-
CoAs. From these observations, we concluded that these
metabolites were acyl-dephospho-CoAs. The representa-
tive LC-TripleTOF 5600+ data is shown in Fig. 2.
Acyl-CoA and acyl-dephospho-CoA profiled by
LC-MS/MS
After dissolving the acyl-CoAs in 100 ␮l of mobile phase A (2%
acetonitrile in 100 mM ammonium formate, pH 5.0), a 40 ␮l
sample was injected on an Agilent ZORBAX 300SB-C8 column
(100 × 2.1 mm, 3.5 ␮m) protected by a guard column (ZORBAX)
(5 ␮m, 12.5 × 2.1 mm) in a Dionex-UltiMate 3000 LC system. The
temperatures of column oven and autosampler were set at 42 and
5°C, respectively. The LC method was developed at 0.2 ml/min-
ute, and i) started with 100% mobile phase A and 0% mobile
phase B (98% acetonitrile in 5 mM ammonium formate, pH 6.3)
for the initial 2 min, ii) mobile phase B was increased to 60%
within 8 min, iii) then mobile phase B was further increased to
90% in 1 min and maintained at 90% for 19 min, and iv) the
gradient was finally returned to the initial condition within 1 min
and the column was reequilibrated with the initial condition for
10 min before the next injection.
The liquid chromatograph was coupled to a 4000 QTRAP mass
spectrometer (Applied Biosystems, Foster City, CA) operated un-
der positive ionization mode with the following source settings:
turbo-ion-spray source at 500°C under N2 nebulization at 65 psi,
N2 heater gas at 55 psi, curtain gas at 30 psi, collision-activated
dissociation gas pressure was held at high, turbo ion-spray voltage
at 5,500 V, DP at 90 V, entrance potential at 10 V, CE at 50 V, and
collision cell exit potential at 10 V. There was no detectable acyl-
CoA located at m/z between 769 and 809 (m/z from free CoA to
acetyl-CoA), thus we skipped precursor ion (Q1) from 770 to
807. This allowed us to set up the MRM method with the Q1 ions
from 768 to 1,100, and their corresponding product ion (Q3)
ions as the subtraction of the Q1 ions by 507. Multiple fragments
of some acyl-CoAs, such as methylmalonyl-CoA, were added to
the MRM transitions to make sure the separation of isobaric acyl-
CoAs. In a separate acyl-dephospho-CoA MRM method, the Q1
ions started from 688 to 988, with the Q3 ions as the subtraction
of their corresponding Q1 ions by 427. Analyst software (version
1.4.2; Applied Biology) was used for data registration.
The detailed fragmentation patterns of acyl-dephospho-
CoAs are outlined in Fig. 1. Acyl-dephospho-CoAs dis-
played the same fragmentation profile as acyl-CoAs assayed
by electrospray ionization-MS/MS. The absence of a phos-
phate group at the 3′-ribose moiety of acyl-dephospho-
CoAs leads to a neutral loss of 427 rather than 507 for
acyl-CoAs, and the other common fragment (5′-phospho-
adenosine) of acyl-dephospho-CoAs becomes m/z 348 cor-
responding to m/z 428 for acyl-CoA.
Method validation
The method was validated using commercially available acyl-
CoAs and dephospho-CoAs in terms of assay linearity, sensitivity,
precision, and accuracy. Sensitivity of the assays was determined
in terms of limits of detection (LODs) and limits of quantitation
(LOQs), which were evaluated by series dilution of standards.
LODs and LOQs are determined as the concentrations which
yield the peak with 3 S/N (signal/noise) and 10 S/N, respec-
tively. Accuracy was determined by a process of spiking of analytes
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Fig. 1. The fragmentation of acyl-CoAs and acyl-dephospho-CoAs in MS/MS. The 3′-phospho group is the only difference between acyl-
CoAs and acyl-dephospho-CoAs, and this difference is reflected by the mass difference of 80 in neutral loss and fragment as well.
To further verify the identity of these CoA analogs,
dephospho-CoA standard and two other acyl-dephospho-
CoAs (acetyl-dephospho-CoA and butyryl-dephospho-CoA)
were synthesized in vitro from dephospho-CoA by acetyl-
CoA synthetase. The resulting data confirmed the identi-
ties of acyl-dephospho-CoAs (supplementary Fig. I).
HPLC separation of free CoA and all acyl-CoAs ranging
from free CoA to C22 was investigated on several reversed
phase columns, mobile phases, and gradient elution pro-
files in order to define optimal assay conditions. Long-
chain acyl-CoAs have very broadened peak shapes, if all
acyl-CoAs including short-chain acyl-CoAs need to be in-
cluded in one method. A high percentage of organic sol-
vent during the initial conditions would be expected to
improve the broadening peaks of long-chain acyl-CoAs.
However, some isobaric acyl-CoAs (short-chain length)
would then coelute under these conditions, such as malonyl-
CoA, 3-hydroxybutyryl-CoA, methylmalonyl-CoA, and suc-
cinyl-CoA. A C8 reversed phase column was found to be
able to reasonably separate all acyl-CoAs from short- to
long-chain length. Increasing ammonium formate in mo-
bile phase A greatly improved peak tailing (though the
maximal concentration achieved was solubility-limited);
the final concentration of ammonium formate employed
was 100 mM in mobile phase A and 5 mM in mobile phase
B. The pH of mobile phase A found to be optimal at 5.0,
used a column with oven temperature at 42°C.
The incidence of peak overlap was also addressed, as
methylmalonyl-CoA is a structural isomer of succinyl-CoA.
Succinyl-CoA is present in much higher concentrations
than methylmalonyl-CoA, so a methylmalonyl-CoA-specific
fragment at m/z 317 was used as a means to ensure selec-
tive quantitation. Under the optimized LC conditions,
acyl-dephospho-CoAs were well-separated with retention
times lagging 1–2 min behind the corresponding acyl-
CoAs due to the loss of a polar phosphate group.
The calibration curve, linearity, and sensitivity (LODs
and LOQs) of acyl-CoAs and dephospho-CoAs are shown
Acyl-(dephospho)-CoA profile by LC-MS/MS
595

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Fig. 2. Extracted data of free CoA, dephospho-CoA, acetyl-CoA, and acetyl-dephospho-CoA in the heart analyzed by MicroLC-TripleTOF
5600+. Extracted LC chromatograms, TOF mass scans, and SWATH fragmentation patterns for free CoA (first row), dephospho-CoA (sec-
ond row), acetyl-CoA (third row), and acetyl-dephospho-CoA (fourth row). The details of the experiments are described in the Methods
section.
in Table 1. All acyl-CoAs and dephospho-CoAs show good
linear signal response across the concentration ranges of
the acyl-CoAs assessed. The programed MRM method is
very sensitive to short- and medium-chain length acyl-CoAs
and dephospho-CoAs. The lower sensitivity to long-chain
acyl-CoAs is likely due to the slight peak broadening and/
or low ionization yield. All the measured standard acyl-
CoAs had some noticeable degradation over the time at
4°C in the autosampler. However, normalized to the inter-
nal standard, the relative standard deviations (RSDs) of
continuous measurements over 27 h for acyl-CoAs at 4°C
in the autosampler were from 11.1 to 23.3%.
extracts. Diluted heart extracts (which possessed no en-
dogenous acyl-CoAs or acyl-dephospho-CoAs) were spiked
with different quantities of acyl-CoAs and dephospho-
CoAs (very low, low, medium, and high concentrations;
the amounts of each compound at different quantities
are listed in supplementary Table I).The choice of spiked
quantities was based on the concentrations of acyl-CoAs
found in real samples. Spiked samples were analyzed and
data are presented in Table 2. The results in Table 2
indicate that the developed MRM method possesses
high accuracy for both acyl- and dephospho-CoAs. The
RSDs of assays at all concentrations of acyl-CoAs were
below 15%, which shows the high precision of the
method.
Methodological accuracy was evaluated by measuring
the known amount of acyl-CoAs spiked into diluted heart
TABLE 1. LODs, LOQs, and calibration curves of LC-MS/MS quantitation method for acyl-CoAs
Linearity
R2
LOD (nM)
LOQ (nM)
Free CoA
Acetyl-CoA
0.9993
0.9919
0.9957
0.9958
0.9954
0.9940
0.9962
0.9998
0.9998
0.9993
0.9944
20
6
10
20
3
60
20
71
133
100
2
63
13
25
50
5
200
60
250
333
333
7
y = 0.4x Ϫ 0.009
y = 1.4x + 0.1
y = 3.5x Ϫ 0.1
y = 0.1x + 0.0005
y = 0.6x Ϫ 0.1
y = 0.9x + 0.02
y = 1.6x + 0.1
y = 1.5x + 0.2
y = 0.6x + 0.1
y = 0.8x + 0.1
y = 1.9x + 0.1
Propionyl-CoA
Malonyl-CoA
Succinyl-CoA
Methylmalonyl-CoA
Octanoyl-CoA
Palmitoyl-CoA
C18:2-CoA
C20:4-CoA
Dephospho-CoA
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TABLE 2. Accuracy of LC-MS/MS method by comparing the measured acyl-CoAs to the spiked amounts at different concentrations
Very Low
Low
Medium
High
Free CoA
98 10
100 14
93 12
104
6
81 10
98 12
108
7
Acetyl-CoA
105 12
97 11
Propionyl-CoA
Succinyl-CoA
Malonyl-CoA
Methylmalonyl-CoA
Octanoyl-CoA
Palmitoyl-CoA
C18:2-CoA
95
114
81
87
97
89 11
91 14
99
81
9
6
6
6
8
100
91
80
3
7
4
7
93
107
91 12
96
101 10
82 11
96
111
105 13
6
3
108
83 10
84
91 10
8
9
95
9
105 10
97 11
94 13
98 10
91 14
99
92
9
7
5
4
C20:4-CoA
Dephospho-CoA
93 10
97 12
4
5
Data in the table are expressed as: assayed concentration/spiked concentration × 100% standard deviation.
Acyl-CoAs possess structural diversity to include satu-
and HMG-CoA) in rat hearts, kidneys, livers, and brains.
Not all saturated acyl-CoAs have the standards. Therefore,
identification of some saturated acyl-CoAs was based on
the measured mass, fragmentation pattern, and chromato-
graphic properties (extracted chromatograms of saturated
acyl-CoAs from various tissues are shown in supplementary
Fig. II). The highest amounts of free CoA and acetyl-CoA
are in the kidney and liver (ف30 nmol/g wet weight). In-
terestingly, the kidney has relatively higher amounts of
other short- and medium-chain saturated acyl-CoAs (C3 to
C10) compared with other organs tested. Odd carbon num-
ber acyl-CoAs, such as heptanoyl-CoA and pentanoyl-CoA,
are uncommon acyl-CoAs. Surprisingly, they were found
to be present in relatively high amounts in the kidney. The
highest amount of propionyl-CoA was also found in the
kidney. Saturated acyl-CoAs with a carbon chain length
>C10 were in the highest abundance in the heart. The brain
rated, unsaturated, hydroxylated, and dicarboxyl functional
groups in the acyl chain. The concentrations of acyl-CoAs
range from trace amounts to relatively high abundance
depending on the specific acyl-CoA and the tissue type of
interest. In general, liver was determined to contain the
greatest quantity of total acyl-CoAs (Fig. 3), and brain the
lowest (Fig. 3).
To clearly visualize and compare the fingerprint of acyl-
CoAs across tissue types, we mapped four categories of
acyl-CoAs in rat heart, kidney, liver, and brain by construct-
ing heat maps. The data in the heat maps are from the
average concentration (nmol/g wet weight) of acyl-CoAs
in each organ assayed. The RSDs of all measured acyl-CoAs
in various organs is not shown in the heat maps, but was
<20% for all assays (n = 3–4). The concentrations of acyl-
CoAs for which direct standards could not be obtained
were calculated based on the calibration curve of a suit-
able standard acyl-CoA which was in close proximity on
the chromatogram.
Figure 4A shows the distribution of saturated acyl-CoAs
(from free CoA to C18-CoA, including methylmalonyl-CoA
Fig. 4. The distributions of saturated acyl-CoAs and unsaturated
acyl-CoAs in rat heart, kidney, liver, and brain (n = 3–4). Red and
black colors represent high and low concentrations, respectively, of
measured acyl-CoAs. A: Saturated acyl-CoA, concentration range is
from 0 to 33.1 nmol/g. B: Unsaturated acyl-CoA, concentration
range is from 0 to 22.9 nmol/g.
Fig. 3. Total acyl-CoAs (nmol/g wet weight) found in rat heart, kid-
ney, liver, and brain. The total amount of acyl-CoAs is the sum of all
measured acyl-CoAs by LC-MS/MS. The significant difference of total
acyl-CoAs between each two organs is indicated by ** (P < 0.05).
Acyl-(dephospho)-CoA profile by LC-MS/MS
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also had relatively high concentrations of C16-CoA and C18-
3-Hydroxyacyl-CoAs are ␤ oxidation intermediates of
fatty acids. 3-Hydroxyacyl-CoAs from various tissues were
identified according to standard and their retention time
(supplementary Fig. IV). The hydroxyl group leads 3-
hydroxyacyl-CoAs to be more polar compared with their
corresponding acyl-CoAs and mono-unsaturated acyl-
CoAs, and this can be demonstrated by their retention
times (supplementary Fig. IV). Cn-3-hydroxyacyl-CoA has
the same mass and mass transition as Cn-1-dicarboxyl-CoA,
this leads to more than one peak in an extracted chro-
matogram (supplementary Fig. IV), and it is the same for
dicarboxyl-CoAs (see the section below and supplemen-
tary Fig. V).The concentrations of 3-hydroxyacyl-CoAs in
tissues are mapped in Fig. 5A. Relatively high levels of
3-hydroxyacyl-CoAs, particularly those with a carbon chain
length >10, are found in the heart. Only trace amounts
of 3-hydroxyacyl-CoAs are found in brain samples.
CoA. Methylmalonyl-CoA and HMG-CoA were predomi-
nantly found in the liver, whereas other organs possessed
5- to 10-fold lower quantities of each.
The distribution of unsaturated acyl-CoAs in the rat or-
gans tested is shown in Fig. 4B. The chromatograms for
these unsaturated acyl-CoAs from various tissues can be
found in supplementary Figs. III-1 and III-2. Unsaturated
acyl-CoAs eluted out slightly earlier than their corre-
sponding saturated acyl-CoAs (supplementary Figs. II,
III-1). Unsaturated acyl-CoAs clustered into two groups
according to their concentrations found in organs. The
first group is the long-chain unsaturated acyl-CoAs, such
as C18:1-, C18:2-, and C20:4-CoA, that are present at high
concentrations in all organs; other long-chain unsatu-
rated acyl-CoAs, like C20:1-, C20:2-, C20:3-, C22:4-, C22:5-,
and C22:6-CoA, mostly locate in the liver and brain. The
second group of unsaturated acyl-CoAs with a carbon
chain length <C16 are below 2 nmol/g wet weight in all
four organs, and nearly undetectable in the brain. The
heart and liver have relatively high levels of unsaturated
acyl-CoAs (C4–C16).
The well-known and small dicarboxyl-CoAs are malonyl-
CoA (C3), succinyl-CoA (C4), and methylmalonyl-CoA (C4).
As expected, malonyl-CoA, succinyl-CoA, and methylmalonyl-
CoA were present in all organs, except for trace amounts
of malonyl-CoA (20- to 30-fold lower) and undetectable
Fig. 5. The distributions of 3-hydroxyacyl-CoAs and dicarboxyl-acyl-CoAs in rat heart, kidney, liver, and brain. Red and black colors rep-
resent high and low concentrations, respectively, of measured acyl-CoAs. The concentration data of acyl-CoAs is the average of 3–4 rats. A:
3-Hydroxyacyl-CoAs, concentration range is from 0 to 2.58 nmol/g. B: Dicarboxyl-acyl-CoAs, concentration range is from 0 to 8.97 nmol/g.
The distribution of dicarboxyl-acyl-CoAs from C5 to C12 is zoomed in the circled insert, concentration range of the insert is from 0 to 0.22
nmol/g.
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methylmalonyl-CoA in the brain (Figs. 4A, 5B). Surpris-
CoA/acetyl-CoA were calculated and are shown in Fig. 7.
Both the brain and kidney had higher dephosphorylation
ratios compared with the heart and liver.
ingly, both the kidney and liver had dicarboxyl-CoAs (C6–
C12), but they were not found in the heart or brain. In
addition, the kidney had relatively higher levels of these
medium-chain dicarboxyl-CoAs compared with the liver.
The extracted chromatograms of the measured dicarboxyl-
CoAs from various tissues are shown in supplementary
Fig. V. The relatively short retention times of dicarboxyl-
CoAs compared with their corresponding acyl-CoAs, mono-
unsaturated acyl-CoAs, and 3-hydroxyacyl-CoAs are due
to the polar carboxylic group.
Figure 6 shows the acyl-CoA compositions of rat organs ac-
cording to structural characteristics (i.e., saturated acyl-CoA,
unsaturated acyl-CoA, hydroxyacyl-CoA, and dicarboxyl-acyl-
CoA). Noteworthy is that succinyl-CoA, methylmalonyl-CoA,
and malonyl-CoA are related to the citric acid cycle, amino
acid metabolism, and fatty acid synthesis, and they are differ-
ent from other dicarboxyl-acyl-CoAs resulting from omega
oxidation. Therefore, succinyl-CoA, methylmalonyl-CoA,
and malonyl-CoA were included in the saturated acyl-CoA
category. In this way, rat heart has 51% saturated acyl-CoAs,
43% unsaturated acyl-CoAs, 6% hydroxyl-acyl-CoAs, and a
small amount of dicarboxyl-acyl-CoAs (<0.1%); rat kidney
has 70% saturated acyl-CoAs, 25% unsaturated acyl-CoAs, 4%
hydroxyl-acyl-CoAs, and 1% dicarboxyl-CoAs; and rat liver
has 54% saturated acyl-CoAs, 44% unsaturated acyl-CoAs, 1%
hydroxyl-acyl-CoAs, and less than 1% dicarboxyl-CoAs. The
brain is mainly comprised of saturated acyl-CoAs (22%)
and unsaturated acyl-CoAs (77%). Minimum quantities
of hydroxyl-acyl-CoAs (<1%) are found in the brain and
dicarboxyl-acyl-CoAs are minimal (<0.1%).
DISCUSSION
MS is the most sensitive method for metabolomic stud-
ies. The development of MRM improves the analytical sen-
sitivity of this approach by allowing the selection of both
precursor and product ions. However, MRM is most com-
monly applied in the quantitation of a targeted compound.
In the current study, the development of a MRM method
based on the fragmentation pattern was successfully used
to quantitatively profile both known and unknown acyl-
CoAs and acyl-dephospho-CoAs in various rat organs with
high sensitivity and accuracy. In total, 67 acyl-CoAs and
acyl-dephospho-CoAs were characterized in the organs
tested, and many acyl-CoAs and acyl-dephospho-CoAs lack-
ing compound authentication were identified by their
mass, fragmentation (loss of 507 for acyl-CoAs or 427 for
acyl-dephospho-CoAs), and chromatographic retention
time. Our results are in agreement with previously re-
ported values (24), suggesting the untargeted approach by
MRM accurately reflects quantities across the range of
carbon chain lengths of physiologically relevant acyl-CoAs.
Furthermore, the relative profiles of acyl-CoAs and acyl-
dephospho-CoAs are in-line with the well-established met-
abolic function and demand in the respective tissues assayed.
Liver tissue has high metabolic activity related to path-
ways, such as fatty acid oxidation, lipid synthesis, glucose
oxidation, gluconeogenesis, and xenobiotic detoxifica-
tion, which is in-line with the finding that liver contains
the highest quantity of total acyl-CoAs (Fig. 3). Although
the unsaturated acyl-CoAs and hydroxyacyl-CoAs found
in the present work cannot exclude other possible isomers,
The acyl-dephospho-CoAs in four rat organs are listed
in Table 3. The highest levels of acyl-dephospho-CoAs
were found in the kidney. Only dephospho-CoA and acetyl-
dephospho-CoA were detected in all four organs. The
ratios of dephospho-CoA/free CoA and acetyl-dephospho-
Fig. 6. The composition of acyl-CoA in each rat or-
gan. The sum of each category of acyl-CoAs composes
the pie figure of each organ. Methyl-malonyl-CoA
and succinyl-CoA were calculated as saturated acyl-
CoAs.
Acyl-(dephospho)-CoA profile by LC-MS/MS
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TABLE 3. Acyl-dephospho-CoA found in rat organs
Acyl-dephospho-CoAs
Heart (nmol/g)
Kidney (nmol/g)
Liver (nmol/g)
Brain (nmol/g)
Dephospho-CoA
0.01 0.01
0.2 0.003
ND
0.02 0.007
ND
ND
ND
0.01 0.002
0.005 0.002
0.003 0.002
4.3 0.8
3.2 0.09
1.4 0.3
0.5 0.02
0.6 0.05
0.3 0.07
0.04 0.01
0.4 0.02
0.09 0.02
0.1 0.03
0.5 0.03
1.0 0.02
0.1 0.01
1.0 0.09
0.02 0.01
0.01 0.01
ND
C2-dephospho-CoA
C3-dephospho-CoA
C4-dephospho-CoA
C5-dephospho-CoA
C6-dephospho-CoA
C7-dephospho-CoA
C8-dephospho-CoA
Succinyl-dephospho-CoA
Malonyl-dephospho-CoA
0.04 0.01
0.02 0.008
0.004 0.005
0.03 0.003
ND
0.04 0.0009
0.04 0.009
0.01 0.004
ND
ND
ND
ND
ND
n = 3ف4. ND, not detected.
enoyl-CoAs and 3-hydroxyacyl-CoAs are major physiologi-
cal metabolites. And with the sample purification, neutral
loss scan, and retention time, this method possesses good
confidence of acyl-CoA and acyl-dephospho-CoA identifi-
cation. However, interferences (non-acyl-CoAs or nonacyl-
dephospho-CoAs) with similar characteristics still cannot
be excluded. Therefore, caution should be taken in iden-
tifying acyl-CoAs with this method in certain circumstances,
such as acyl-CoA metabolites from xenobiotics or drugs. In
such cases, further characterization is needed to confirm
their identities, such as using fragment ion scans, high
resolution MS, isotope labeling, or authentic compounds.
The heart is a high energy demand organ where fatty
acids serve as the preferred energy fuel for ATP produc-
tion. This metabolic phenotype is reflected in our mapped
data: i) several saturated acyl-CoAs (C10-, C12-, C14-, and C18-
CoAs) are the highest in heart; ii) the corresponding un-
saturated acyl-CoAs and 3-hydroxyacyl-CoAs, i.e., C10-, C12-,
C14-, and C18-CoAs, are also relatively high in the heart.
Saturated acyl-CoAs, enoyl-CoAs, and 3-hydroxyacyl-CoAs are
␤ oxidation intermediates of fatty acids, the high amounts
of these acyl-CoAs reflect the high activity of fatty acid oxi-
dation in the heart.
Interestingly, high amounts of short- and medium-chain
saturated acyl-CoAs (C3–C10) were present in the kidney.
This is possibly due to reabsorption of acyl-carnitines by
the kidney and further metabolism for energy production
in the kidney. Odd chain saturated acyl-CoAs (heptanoyl-
CoA and pentanoyl-CoA) were also found in particularly
high amounts in kidney tissue and do not appear to come
from the ␤ oxidation of long-chain fatty acids with odd
carbon numbers because longer chain acyl-CoAs (C > 7)
with odd carbon numbers were not detected. We also ob-
served that the kidney contained the highest amount of
propionyl-CoA, a downstream metabolite of both pentanoyl-
CoA and heptanoyl-CoA. Propionyl-CoA is an anaplerotic
substrate that enters the citric acid cycle via carboxylation
and isomerization to methylmalonyl-CoA and succinyl-CoA,
respectively (31, 32). However, methylmalonyl-CoA and
succinyl-CoA are not equivalently high in the kidney;
10-fold less, and one-half that of liver, respectively. This
mismatched concentration of methylmalonyl-CoA or suc-
cinyl-CoA may be linked to the lower amount of ATP in
the kidney, as ATP is involved in the carboxylation of pro-
pionyl-CoA to methylmalonyl-CoA.
The brain prefers glucose or ketone bodies as energy
fuel and ␤ oxidation of fatty acids is a minor contribution.
This was reflected in the acyl-CoA fingerprint generated
with our methodological approach. The largest portion of
acyl-CoAs is long-chain saturated and unsaturated acyl-
CoAs (C16 or above), representing ف86% of total acyl-CoAs
in the rat brain. Long-chain unsaturated fatty acids, such
as oleic acid and arachidonic acid, are largely used for
phospholipid synthesis and must be activated to acyl-CoAs
before being used in the synthesis of phospholipids for in-
corporation into membranes (33, 34). The high abun-
dance long-chain acyl-CoAs and diminutive amounts of
other acyl-CoAs strongly indicate low ␤ oxidation and high
lipid synthesis, particularly phospholipids, in the brain.
Beta oxidation intermediates, such as enoyl-CoAs and
3-hydroxyacyl-CoAs, were negligible in the brain.
Dicarboxyl-CoAs, except for malonyl-CoA, succinyl-CoA,
and methylmalonyl-CoA, are metabolites of omega oxida-
tion of medium-chain or long-chain fatty acids. Omega
oxidation of fatty acids is initiated from omega hydroxy-
lation by P450 enzymes at the terminal (omega) carbon
of the fatty acid (35). We have recently observed that the
kidney and liver have high omega hydroxylation activity,
Fig. 7. The ratios of dephospho-CoA/free CoA and
acetyl-dephospho-CoA/acetyl-CoA in rat heart, kid-
ney, liver, and brain. Left panel: the ratio of dephos-
pho-CoA/free CoA found in four organs. Right panel:
the ratio of acetyl-dephospho-CoA/acetyl-CoA found
in four organs. The significant difference of ratios be-
tween each two organs is indicated by ** (P < 0.05).
600
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8. Zhang, G. F., S. Sadhukhan, R. A. Ibarra, S. M. Lauden, C. Y.
whereas the heart and brain are very low (Q. Li et al., un-
published observations). This is consistent with the re-
latively high quantities of short- and medium-chain
dicarboxyl-acyl-CoAs in the liver and kidney and the negli-
gible amounts in the heart and brain in this study.
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Summarized from the acyl-CoA fingerprint in four or-
gans as shown in the pie figure (Fig. 6), enoyl-CoAs (from
C4–C16) and hydroxyl-CoAs in the heart indicate high fatty
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minutes in the ischemic brain (37).
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range of cellular acyl-CoAs including characterization of a
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