Quantification and molecular imaging of fatty acid isomers from complex biological samples by mass spectrometry

Article abstract of DOI:10.1039/d1sc01614h

Elucidating the isomeric structure of free fatty acids (FAs) in biological samples is essential to comprehend their biological functions in various physiological and pathological processes. Herein, we report a novel approach of using peracetic acid (PAA) induced epoxidation coupled with mass spectrometry (MS) for localization of the CC bond in unsaturated FAs, which enables both quantification and spatial visualization of FA isomers from biological samples. Abundant diagnostic fragment ions indicative of the CC positions were produced upon fragmentation of the FA epoxides derived from either in-solution or on-tissue PAA epoxidation of free FAs. The performance of the proposed approach was evaluated by analysis of FAs in human cell lines as well as mapping the FA isomers from cancer tissue samples with MALDI-TOF/TOF-MS. Merits of the newly developed method include high sensitivity, simplicity, high reaction efficiency, and capability of spatial characterization of FA isomers in tissue samples.

Full text of DOI:10.1039/d1sc01614h

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Quantification and molecular imaging of fatty acid
isomers from complex biological samples by mass
spectrometry†
Cite this: Chem. Sci., 2021, 12, 8115
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
a
b
a
b
Hua Zhang,
Meng Xu,
Xudong Shi,c Yuan Liu,
Zihui Li,
Justin C. Jagodinsky,d Min Ma,a Nathan V. Welham,
Zachary S. Morrisd
c
ab
*
and Lingjun Li
Elucidating the isomeric structure of free fatty acids (FAs) in biological samples is essential to comprehend
their biological functions in various physiological and pathological processes. Herein, we report a novel
approach of using peracetic acid (PAA) induced epoxidation coupled with mass spectrometry (MS) for
localization of the C]C bond in unsaturated FAs, which enables both quantification and spatial
visualization of FA isomers from biological samples. Abundant diagnostic fragment ions indicative of the
C]C positions were produced upon fragmentation of the FA epoxides derived from either in-solution or
on-tissue PAA epoxidation of free FAs. The performance of the proposed approach was evaluated by
analysis of FAs in human cell lines as well as mapping the FA isomers from cancer tissue samples with
MALDI-TOF/TOF-MS. Merits of the newly developed method include high sensitivity, simplicity, high
reaction efficiency, and capability of spatial characterization of FA isomers in tissue samples.
Received 20th March 2021
Accepted 4th May 2021
DOI: 10.1039/d1sc01614h
rsc.li/chemical-science
ne structures of FA C]C bond positional isomers. Conven-
tional low-energy collisional activation methods, such as
Introduction
collision-induced-dissociation (CID) tandem MS, are ineffective
in recognizing the C]C bond positions in FA isomers due to the
high energy required to cleave the C]C bond.11,12 To circum-
vent this limitation, extensive efforts have been devoted to
achieve unambiguous identication of FA isomers from bio-
logical samples in recent years.13–17 For instance, alternative ion
activation methods14,16,18–22 have been explored to determine
lipid isomers owing to the great advancements in instrumen-
tation. However, these methods usually require specic MS
instrument modications or specialized instrumentation.
Moreover, cleaving the C]C bond with high fragmentation
energy is usually accompanied by the breakage of carbon–
carbon single bonds, which increases the complexity of the
spectra, and lowers the abundance of diagnostic ions.18,23
Another emerging strategy to locate the C]C bond position
is implementation of a C]C derivatization procedure prior to
Structural diversity and varying expression levels of fatty acids
(FAs) are closely related to their unique roles in cell metabolic
pathways. Positional isomers of FA with different carbon–
carbon double bond (C]C) positions possess considerably
different roles in various biological functions.1–3 Metabolic
dysregulations of FA C]C bond positional isomers may
contribute to the pathogenesis of cancer,4 cardiovascular
diseases,5 Alzheimer's and other neurodegenerative diseases.6
Therefore, precise elucidation of FA isomeric structures in
biological samples is critical for a deeper understanding of their
biological functions.
Mass spectrometry (MS) is emerging as one of the most
informative techniques for the study of FAs due to its superior
analytical power.7–9 Although signicant advancements have
occurred in lipidomics research using various MS techniques
including shotgun MS10 and hyphenated chromatographic MS
techniques (LC/GC-MS),7 challenges remain in elucidating the
CID MS/MS interrogation. C]C bond chemical derivatization
13,15,24,25
`
¨
methods based on the Paterno–Buchi (PB) reactions,
ozonolysis,26,27 meta-chloroperoxybenzoic acid (m-CPBA)-epoxi-
dation,28,29 and plasma/electrochemical induced epoxida-
tion,30–33 have been utilized to induce structurally informative
dissociation of FAs in CID MS/MS in recent years. Additionally,
covalent adduct chemical ionization (CACI) GC MS/MS34 and
gas-phase ion/ion reactions35 have been adopted for the char-
acterization of isomeric FAs. Nevertheless, most of these
approaches are performed under in-solution (bulk) conditions,
therefore sacricing FA spatial information. Visualization of FA
aSchool of Pharmacy, University of Wisconsin–Madison, Madison, WI, 53705, USA.
E-mail: lingjun.li@wisc.edu
bDepartment of Chemistry, University of Wisconsin–Madison, Madison, WI, 53706,
USA
cDivision of Otolaryngology, Department of Surgery, School of Medicine and Public
Health, University of Wisconsin–Madison, Madison, WI, 53792, USA
dDepartment of Human Oncology, School of Medicine and Public Health, University of
Wisconsin–Madison, Madison, WI, 53705, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d1sc01614h
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isomers across tissue samples provides critical information to C]C bond was readily cleaved yielding an aldehyde and an
better understand their region-specic functions. It is noted olen fragment ion pair. Affirmative C]C bond positional
15,17,36
`
¨
that the Paterno–Buchi reaction,
ozone-induced dissocia- information was then deduced from the characteristic fragment
tion (OzID),16 ozonolysis37 and m-CPBA epoxidation29 have been ion pair.
adopted for MS-based mapping of lipid isomers on tissues,
To test the feasibility of this approach, in-solution epoxida-
however, challenges still remain including low yields, side tion was conducted using unsaturated FA standards upon
reactions, and interference from excess on-tissue derivatization reaction with PAA and the epoxide products were subjected to
reagent. Therefore, the development of novel methodologies for nanoESI-MS/MS analysis. The unsaturated FAs were completely
spatial mapping and quantication of FA isomers within bio- converted into their epoxy forms aer the PAA treatment
logical samples is still in great demand.
(Fig. S2†). To further conrm the structure of these epoxide
Herein, we report a novel approach of using peracetic acid products, tandem MS experiments were performed. As shown in
(PAA)-induced epoxidation in MS for localization of C]C bond Fig. 2, abundant diagnostic fragment ion pairs indicative of the
in unsaturated FAs, which enables both quantication and C]C positions were obtained in the CID MS/MS. Additionally,
spatial visualization of FA isomers from biological samples HCD tandem MS was performed for the interrogation of these
without need for instrumental modications and can be epoxide products using varied normalized collision energy
broadly transferrable to various MS platforms. The performance (NCE) (Fig. S3 and S4†). Although diagnostic fragment ions
of the proposed approach was demonstrated by analysis of FA indicating the C]C were observed using both fragmentation
isomer standards, FAs in HPV16-E6E7 immortalized human methods, diagnostic fragment ions with relatively higher
pancreatic duct epithelial (HPDE/E6E7) cells and pancreatic abundance, as well as cleaner mass spectra, were obtained
cancer cells (PANC-1) using shotgun MS, as well as mapping of using CID mode at NCE of 35%, presumably due to preferential
FA isomers from cancer tissues based on a matrix-assisted laser cleavage of the three-membered oxirane ring structure.
desorption/ionization time-of-ight/time-of-ight MS (MALDI- Furthermore, polyunsaturated FAs including FA 18:3 (6Z, 9Z,
TOF/TOF-MS) platform. In these experiments, FA isomers 12Z) and FA 20:4 (5Z, 8Z, 12Z, 14Z) were analyzed with their
were unambiguously and sensitively identied and quantied. C]C bond positions deduced from their diagnostic fragments,
Additionally, the heterogeneity of FA isomers was mapped on despite having more complex MS/MS spectra (Fig. S5†).
tumor tissue sections from a murine tumor model. Overall, our
results indicate that the PAA epoxidation coupling with MS
approach provides a versatile platform for elucidating the ne
structures of FAs, which help to better understand their physi-
ological functions.
Quantitative analysis of C]C bond positional FA isomers
The analytical performance of the proposed method was further
examined by quantitatively investigating FAs with different
C]C bond locations. As shown in Fig. S6,† satisfactory linearity
was attained for FA 16:1 (9Z), FA 18:1 (9Z), and FA 18:1 (11Z) over
a dynamic range of 0.05–10.0 mM, with R2 values > 0.9989. The
limit of detection (LOD), dened by S/N ¼ 3, was estimated to be
as low as 4.4 nM for FA 16:1 (9Z), 5.6 nM for FA 18:1 (9Z), and
4.2 nM for FA 18:1 (11Z). The relative standard deviations
(RSDs) (n ¼ 3) from all the examined samples were below 5.5%,
3.4%, and 8.9% for FA 16:1 (9Z), FA 18:1 (9Z), and FA 18:1 (11Z),
respectively. These data show that the proposed method
exhibits higher sensitivity for the detection of FA isomers than
recently reported methods,24,32,33 which attributes to the high
labeling efficiency and high yield of diagnostic fragment ions.
Relative quantication of FA isomers was also performed by
analyzing a series of mixture solutions of FA 18:1 (9Z) and FA
18:1 (11Z), with varied molar ratios from 0.001 : 1 to 10 : 1, at
a total concentration of 35 mM. Diagnostic fragment ion pairs
from FA 18:1 (9Z) and FA 18:1 (11Z) were observed from the MS/
MS spectrum of the isomer mixture (Fig. S7†). As illustrated in
Fig. S6d,† satisfactory linearity with R2 ¼ 0.997 was achieved
over a dynamic range of molar ratios from 0.001 : 1 to 10 : 1.
The RSDs of three replicates for all the examined samples were
consistently below 6%.
Results and discussion
Localization C]C bond in FA by PAA epoxidation coupled
with tandem MS
The mechanism of pinpointing C]C bond in FAs by PAA-
initiated epoxidation and tandem MS is shown in Fig. 1. The
unsaturated FAs were converted into FA epoxides aer the
treatment of PAA under either in-solution reaction or on-tissue
epoxidation using the vapor of PAA (Fig. S1†). The epoxy-FAs
were subsequently subjected to tandem MS fragmentation,
under which the three-membered oxirane ring derived from the
Fig. 1 PAA epoxidation used in conjunction with MS for pinpointing Analysis of unsaturated FAs in cell-line samples
C]C bond in FAs. (a) Schematic illustration of pinpointing C]C
To test the applicability of the proposed method in complex
position in unsaturated FAs using PAA epoxidation coupled with
biological samples, FAs were extracted from human pancreatic
duct epithelial cells (HPDE cells), pancreatic cancer cells (PANC-
tandem MS. MS interrogation method for (b) bulk samples and (c) MS
imaging of tissue sections.
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Fig. 2 MS/MS spectra of FA epoxides after the in-solution epoxidation of unsaturated FAs: (a) FA 16:1 (9Z), (b) FA 18:1 (6Z), (c) FA 20:1 (11Z), (d) FA
18:1 (9Z), (e) FA 18:2 (9Z, 12Z), (f) FA 18:1 (11Z).
1 cells), and PANC-1 cells treated with stearoyl-CoA desaturase 1
(SCD1) inhibitor A939572 38 – a drug that inhibits conversion of
saturated fatty acyl-CoA to monounsaturated fatty acyl-CoAs.
Following PAA epoxidation, FA samples were subjected to
nanoESI-MS/MS analysis. A total of 37 monounsaturated FAs
with denitive C]C bond positions were identied (Table S1†),
showing a high heterogeneity of the FA isomeric composition in
these pancreatic cell samples. Interestingly, the proportions of
different FA isomers exhibited signicant variation between the
HPDE and PANC-1 cells (Fig. 3). FA 16:1 (D9) was observed as
the major isomer for FA 16:1 in both the HPDE and PANC-1
cells, while its proportion decreased from 80.7 ꢀ 1.9% for
HPDE cells to 33.1 ꢀ 6.5% for PANC-1 cells (Fig. 3a). A similar
decrease in proportion was found for the major isomers of FA
18:1 (D9) and FA 18:1 (D11) in PANC-1 cells (Fig. 3b). In contrast,
increased proportions of some low abundance isomers were
observed for FA 16: 1, FA 18:1 and FA 20:1 in the PANC-1 cells.
These ndings suggest that important FA metabolic pathway
alterations may exist in the cancer cell line compared to its
normal counterpart. In our proof-of-principle study, the FAs
isomers from cell-line samples were tentatively assigned based
on high resolution tandem MS data (with tight mass error
within 5 ppm). Note that the condence of isomeric identi-
cation of the low abundance FA isomers could be further
increased by combining with other lipid C]C double bond
characterization methods such as PB reactions,17,24 covalent
adduct chemical ionization (CACI) GC MS/MS,34 ozone-induced
dissociation.19 Unfortunately, these techniques usually require
specic MS instrument modications or specialized instru-
Fig. 3 Analysis of C]C positional isomers for three major mono-
unsaturated FA in pancreatic cells. (a) FA 16:1, (b) FA 18:1, (c) FA 20:1.
The green, red, and blue bars represent data obtained from HPDE cells,
PANC-1 cells, and PANC-1 cells treated with SCD1 inhibitor A939572,
mentation, which are not directly available on our current hi-res
MS instrument. Nonetheless, these methods can be exploited
for more condent FAs isomeric identication in future studies.
Further, PANC-1 cells dosed with 200 nM SCD1 inhibitor
A939572 38 were tentatively investigated. No signicant change
was observed in the isomeric compositions for FA 16:1 and FA
18:1, while several isomers from FA 20:1 were notably different
respectively. Differences between the two groups were evaluated
using the two-tailed Student's t tests, *P < 0.05, **P < 0.01, ***P <
0.001, and ****P < 0.0001. n ¼ 3.
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aer the 24 hour drug treatment.38 Inhibition of SCD1 has been pattern in that lipid signal intensity (FA 18:1 and FA 18:0) is
found to effectively suppress proliferation and induce apoptosis stronger in the peripheral tissue region rich in cancer cells
of tumor cells.38 The limited FA metabolic change in PANC-1 compared to the necrotic inner tissue region that contains fewer
cells aer the drug treatment in the current case might be cancer cells (Fig. 4 a and b). Receiver Operating Characteristic
due to an insufficient drug treatment period, as the 24 hour (ROC) tests of the intensity of FA 18:1 between the radiated and
treatment did not yield a signicant change in cell density (data control tissues resulted in an area under the curve (AUC) value
not shown). It is worth mentioning that although the C]C of 0.647 (Fig. S9†), which indicates a slight decrease in FA 18:1
bond positions could be obtained for polyunsaturated FA aer radiation treatment. We successfully mapped 5 isomers of
standards (Fig. S5†), the in-depth analysis of polyunsaturated FA 18:1 with MALDI-TOF/TOF MS/MS imaging targeted at
FAs in complex biological samples would benet from addi- epoxy-FA 18:1 (Fig. 4 e–i and S10†). One isomer (FA18:1(D9))
tional chromatographic separation29,33 to achieve more accurate showed signicantly decreased signal intensity in the cancer
identication of C]C bond position. A related study is region aer radiation treatment compared to untreated
currently underway in our laboratory.
samples (Fig. 4h and S11†). This demonstrates the biological
signicance of detecting FA isomers on tissue sections.
A segmentation pipeline was employed to segment the MS/
MS data into two distinct regions, which were tentatively iden-
tied as tumor and necrotic regions aer comparing with the
histological image (Fig. 4j and k). Based on the segmentation
and histology results, we found that downregulation of FA 18:1
isomers was tightly associated with tumor necrosis. Specically,
FA 18:1 (D9) yielded AUC value of 0.964 in comparisons between
the tumor and necrotic regions (Fig. S12†), suggesting that the
concentration of FA 18:1 (D9) was notably reduced in necrosis.
This result highlights the potential value of FAs as downstream
biomarkers that reect metabolic alterations linked to the
progression of tumor necrosis.
It is worth mentioning that m-CPBA epoxidation method was
also adopted for pinpointing C]C bond positions in unsatu-
rated lipid recently.28,29 Both the m-CPBA and PAA strategies
have features such as high sensitivity, high specicity, and
simplicity with no need for instrumental modications. In
comparison with the m-CPBA epoxidation, the advances of PAA
epoxidation over the m-CPBA treatment are also obvious. (i) For
in-solution derivatization, it is much easier for sample clean-up
with PAA as the excess PAA in the reaction system could be
Spatial characterization of C]C positional isomers using
MALDI-TOF/TOF-MS/MS
We further applied the PAA epoxidation strategy to map the
spatial distribution of FA isomers within tissue samples using
a
matrix-assisted laser desorption/ionization time-of-ight
tandem mass spectrometry (MALDI-TOF/TOF MS) platform.
MALDI-TOF/TOF MS imaging was performed on tumor tissue
sections from a murine melanoma model. The PAA incubation
time was set to 2 h to ensure complete epoxidation of unsatu-
rated FAs (Fig. S8†). As shown in Fig. 4, the spatial distribution
of FA 18:0 at m/z 283.2, and epoxy-FA 18:1 at m/z 297.2 obtained
from a tissue section pretreated with PAA epoxidation (Fig. 4 c
and d) were in line with that of FA 18:0 at m/z 283.2, and FA 18:1
at m/z 281.2 from an adjacent underivatized tissue section
(Fig. 4 a and b). These results show that the on-tissue epoxi-
dation reaction process has neither dislocated the unreacted
saturated FAs (such as FA 18:0), nor altered the distribution of
reacted FA species, providing accurate spatial distribution of
the target analytes. The untreated control (le side section) and
the radiation-treated tissue (right side) exhibit the same spatial
Fig. 4 Full-MS images of (a) FA 18:1 and (b) FA 18:0 without epoxidation; full-MS images of (c) epoxy-FA 18:1 and (d) FA 18:0 after on-tissue
epoxidation treatment. MALDI-TOF/TOF-tandem MS images (e) FA 18:1 (D6), (f) FA 18:1 (D7), (g) FA 18:1 (D8), (h) FA 18:1 (D9), (i) FA 18:1 (D11). (j)
The H&E stained histological image. (k) Segmentation pipeline analysis of the MS2 data: region rich in cancer cells (red) and region containing few
cancer cells with necrosis (green). The left side tissue section was from untreated sample while the right-side tissue section was from sample
treated with radiation in all the images. Mass tolerance of all the full-MS images is ꢀ0.05 Da whereas that of ꢀ0.1 Da for MS/MS images.
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easily removed through a SpeedVac owing to its high volatility, future to further improve the identication accuracy of poly-
whereas m-CPBA is a nonvolatile reagent which can be difficult unsaturated FAs.
to remove from the sample using a SpeedVac and needs much
more tedious sample preparation steps to clean-up excess m-
CPBA. (ii) For on-tissue epoxidation, a notable advantage of PAA
is minimal chemical contamination. Since PAA rapidly evapo-
Experimental
Materials and reagents
rates from the tissue aer incubation due to its high volatility,
potential side effects such as ion suppression for subsequent
MS imaging analysis are greatly minimized. However, a layer of
m-CPBA would be coated on the tissue aer spraying the m-
CPBA reagent onto the tissue (Fig. S13†). The excess m-CPBA on
the tissue would oxidize the MALDI matrix and suppress the
signal of derivatization products. A comparison between the
PAA and m-CPBA methods using MALDI-TOF/TOF MS/MS
under consistent instrumental parameters showed that the
signal was 6-fold higher in the PAA treated samples compared to
the m-CPBA treated samples at the MS2 level (Fig. S14†). (iii)
Another advance of PAA over m-CPBA is the on-tissue epoxida-
tion found to be much faster when using PAA. It takes less than
2 hours to reach a nearly 100% epoxidation efficiency for the
unsaturated FAs with PAA (Fig. S8†), whereas the m-CPBA
strategy needs overnight incubation (ca. 18 h) in a sealed
humidity chamber to achieve nearly 100% reaction efficiency.
For details about the materials and reagents used in the study,
refer to the ESI.†
Pancreatic cell-lines
The commercial pancreatic cancer cell line PANC-1 (ATCC®
CRL-1469) obtained from American Type Culture Collection
(ATCC, Manassas, VA, USA) was cultured and maintained in
DMEM:F12 (Hyclone, VWR International, Radnor, PA, USA)
containing 10% fetal bovine serum (FBS) (Gibco, Thermo
Fischer, Waltham, MA, USA), 1% penicillin–streptomycin solu-
tion (Gibco, Thermo Fischer Scientic, Waltham, MA, USA). The
HPV16-E6E7 immortalized human pancreatic duct epithelial
(HPDE/E6E7) cell line obtained from Applied Biological Mate-
rials (Richmond, BC, Canada) was maintained in DMEM:F12
containing 10% FBS, 1% penicillin–streptomycin solution. All
cells were cultured in a 37 ꢁC moisture incubator supplied with
5% CO2. The stearoyl-CoA desaturase 1 (SCD1) inhibitor
A939572 was dissolved in DMSO (Sigma-Aldrich, St. Louis, MO)
at a concentration of 10 mM divided to 100 mL aliquots and
Conclusions
ꢁ
stored at ꢂ20 C until use. PANC-1 cells and HPDE cells were
In summary, a versatile approach for the structural characteriza-
tion of FA C]C bond isomers via PAA epoxidation coupled with
tandem MS has been developed in this study, which enables both
quantication and spatial mapping of the distribution of FA
isomers from biological samples. Accurate localization of C]C
bond position in FAs has been achieved based on the character-
istic diagnostic fragments yielded by the epoxidated FAs under
tandem MS conditions such as CID, HCD on an ESI Orbitrap
platform and a MALDI-TOF/TOF tandem MS platform. A PAA
epoxidation reaction of C]C bond from unsaturated FAs can be
seeded to 10 cm cell culture dishes at a density of 1.5 ꢃ 106 cells
per dish then allowed to attach for 24 hours. Aer attachment,
medium was replaced by serum free DMEM:F12. PANC-1 cells
were treated with 200 nM A939572 or an equivalent volume of
DMSO (control group, 1% DMSO) in triplicate. HPDE/E6E7 cells
were treated with an equivalent volume of DMSO. All the cell
samples were harvested aer 24 h in culture.
Murine tumor models
implemented through either in-solution or on-tissue with close to The murine melanoma B78-D14 (B78) cell line, derived from
100% labeling efficiency. The data show that the proposed B16 melanoma as previously described, was obtained from
method exhibits higher sensitivity than recently reported Ralph Reisfeld (Scripps Research Institute) in 2002.39 B78 cells
methods for the detection of FA isomers. Furthermore, our were grown in RPMI-1640 and were supplemented with 10%
method does not need any MS instrumental modication which FBS, 100 U mLꢂ1 penicillin, and 100 mg mLꢂ1 streptomycin.
offers general accessibility as well as compatibility with Cells were incubated in a humidied incubator at 37 ꢁC with 5%
commercial mass spectrometers. Overall, the merits of the newly CO2. Cell line authentication was performed per ATCC guide-
developed method include excellent sensitivity, no MS instrument lines using morphology, growth curves, and mycoplasma
modication requirements, high reaction efficiency, and the testing within 6 months of use.
provision of spatially resolved FA isomeric information on tissue
Mice were housed and treated under a protocol approved by
samples. These advances will facilitate research endeavors for ne the Institutional Animal Care and Use Committee (IACUC) at
structural elucidation and characterization of FAs in structural the University of Wisconsin–Madison. Female C57BL/6 mice
lipidomics. It is noted that, although the PAA epoxidation coupled were purchased at age 6 to 8 weeks from Taconic Biosciences,
with tandem MS strategy is feasible for the identication of both Inc. (Rensselaer, NY, USA.). B78 tumors were engraed by
monounsaturated and polyunsaturated FAs, we note that the PAA subcutaneous ank injection of 2 ꢃ 106 tumor cells. Tumor size
epoxidation method provides easier interpretation for mono- was determined using calipers and volume approximated as
unsaturated FAs than polyunsaturated FAs, as the tandem mass (width2 ꢃ length)/2. Treatment began when tumors were well-
spectra of epoxy-polyunsaturated FAs become more complex. To established (ꢄ500 mm3), which occurred approximately 5
facilitate the identication process, establishing a spectral library weeks following the engrament. Before treatment initiation,
and data searching interface for automated matching and anno- mice were randomized to receive either external beam radiation
tation of the diagnostic fragment ions will be explored in the (EBRT) or no treatment.
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Delivery of EBRT in vivo was performed using an X-ray bio- FA 18:1 (11Z) and FA 16:1 (9Z). To plot the calibration curves,
logical cabinet irradiator X-RAD 320 (Precision X-ray, Inc., North a series of working solutions at 0.01–40.00 mM of FA 18:1 (9Z),
Branford, CT, USA). EBRT was prescribed to 20 Gy. The dose rate FA 18:1 (11Z) and FA 16:1 (9Z) containing a constant 5 mM FA
for EBRT delivery in all experiments was approximately 2 18:1 (6Z) internal standard were subjected to nanoESI-MS/MS
Gy minꢂ1. Dosimetric calibration and monthly quality assur- analysis, respectively. The signal intensities of diagnostic ion
ance checks were performed on these irradiators by University pairs were summed for each specic FAs, and the ratios of the
of Wisconsin Medical Physics Staff. The tumors where har- summed signal intensities between the FA and the IS were
vested 24 hours aer EBRT treatment and stored under ꢂ80 ꢁC plotted against the concentrations of FA. Relative quantication
before use.
of FA isomers was also demonstrated by analyzing a series of
To prepare the tissue section for MS imaging, the tumor mixture solution of FA 18:1 (9Z) and FA 18:1 (11Z) with varied
tissue was embedded in gelatin aqueous solution (100 mg molar ratios from 0.001 : 1 to 10 : 1, at a total concentration of
mLꢂ1), followed by snap frozen on dry ice. The tumor tissue was 35 mM. To plot the relative quantication calibration curve, the
sectioned at 12 mm thickness using a cryostat (ThermoFisher signal intensities of diagnostic fragment ions were summed for
Scientic, San Jose, CA, USA) set at ꢂ20 ꢁC and the tissue FA 18:1 (9Z) and FA 18:1 (11Z), respectively, and the summed
sections were mounted on the ITO-coated glass microscope signal intensity ratios between FA 18:1 (9Z) and FA 18:1 (11Z)
slides. The tissꢁue section samples were dried in a desiccator and were plotted against their molar ratios.
stored at ꢂ80 C until analysis.
On-tissue epoxidation of unsaturated FAs for MALDI-TOF/TOF
In-solution epoxidation of unsaturated FAs for shotgun
lipidomics
MSI
The schematic analytical workow of on-tissue epoxidation of
Unsaturated FA standards including FA 16:1 (9Z), FA 18:1 (6Z), FA FAs from tissue section samples for MALDI-TOF-MSI was shown
18:1 (9Z), FA 18:1 (11Z), FA 20:1 (11Z), FA 18:2 (9Z, 12Z), g-lino- in Fig. S1.† The tissue sections were thawed in a desiccator for
lenic acid FA 18:3 (6Z, 9Z, 12Z), and arachidonic acid FA 18:4 (5Z, 10 min at room temperature. Aer drying, the tissue sections
8Z, 11Z, 14Z) were dissolved in 2-propanol (IPA) at a concentra- were incubated in a sealed chamber with PAA vapor (PAA, 10%
tion of 2 mg mLꢂ1 as stock solutions, respectively. The FAs stock (v/v) aqueous solution) for 2 h. Aerwards, 1,5-diaminonaph-
solutions were stored under ꢂ20 ꢁC before use. PAA solution was thalene (DAN) matrix in acetonitrile/water (v/v, 70 : 30) solution
also prepared in IPA to a concentration of 500 mM. For deriva- at concentration of 10 mg mLꢂ1 was sprayed onto the tissue
tization of FA standards, an aliquot of 20 mL FA standard solution section using a M5-Sprayer (HTX Technologies, Carrboro, NC,
(10 mM) and 20 mL PAA solution (500 mM) were mixed in USA) at a ow rate of 50 mL minꢂ1 with tracking space of 2 mm
a 0.6 mL tube, and then the mixture was shaken at 300 rpm at for 2 passes. The drying time between each pass was set to 30 s.
room temperature for 1 h. Completed epoxidation of unsaturated The nozzle temperature was set to 50 ꢁC, the nozzle nitrogen gas
FAs was readily achieved by adding excessed amount of PAA. pressure was 10 psi, and the moving velocity of the nozzle was
Aerwards, 360 mL IPA was added to dilute the reaction system. 1000 mm minꢂ1. For samples without on-tissue epoxidation,
The extraction of fatty acids from the cell samples used a modi- the PAA incubation was skipped from the workow aforemen-
ed Folch protocol. Briey, cell suspensions were centrifuged at tioned. The tissue section samples were dried inside a desic-
800g for 2 min, aer which the upper aqueous layer was dis- cator for 20 min before MALDI-TOF-MS imaging. In
carded. Then, the cells were washed with 2 mL dPBS for three a comparison experiment of using m-CPBA for on-tissue epox-
times (800g and 2 min centrifugation) and the cell pellets were idation, m-CPBA in methanol/water (v/v, 50 : 50) solution at
obtained. Aerwards, 0.5 mL water, 0.5 mL methanol and 0.5 mL concentration of 10 mg mLꢂ1 was sprayed onto the tissue
chloroform were added into the cell tube. The mixture was section using a M5-Sprayer at a ow rate of 100 mL minꢂ1 with
sonicated in a water bath for 15 min and vigorous vortexing for tracking space of 2 mm for 4 passes. The drying time between
2 min before centrifugation (12 000g) at 4 ꢁC for 10 min. The each pass was set to 10 s. The nozzle temperature was set to
chloroform layer was transferred to a screw-capped glass tube. 30 ꢁC, the nozzle nitrogen gas pressure was 10 psi, and the
The extraction repeated once and the combined chloroform moving velocity of the nozzle was 600 mm minꢂ1. Then, the m-
layers were dried under a nitrogen stream. All the lipid extracts CPBA coated tissue section (Fig. S13†) was incubated in
ꢁ
were stored at ꢂ20 C before analysis. The FAs extract total was a humidity chamber (water in the bottom) under 37 ꢁC for 18 h
reconstituted in 40 mL IPA. The epoxidation was carried out with to reach a high epoxidation efficiency. The application of DAN
20 mL FAs extract and 20 mL PAA (500 mM), aer 1 h shaking (300 matrix and MALDI-TOF-MS parameters were kept consistent
rpm) at room temperature, the reaction system was diluted with with the scenario of using the PAA reagent.
360 mL IPA. All the derivatized samples were further diluted to
target concentration using IPA for nanoESI-MS/MS interrogation
without any other pretreatment.
Data acquisition and analysis
The shotgun nanoESI-MS analysis was carried out on Orbitrap
Fusion™ Lumos™ Tribrid™ mass spectrometer (Thermo
Scientic, San Jose, CA, USA). Mass spectra were collected in
Quantitative analysis
For absolute quantication of FA isomers, FA 18:1 (6Z) was used a mass range of m/z 100–600 in negative ion detection mode.
as internal standard (IS) for quantitative analysis of FA 18:1 (9Z), The ionization voltage was set to ꢂ2.0 kV. The heated ion
8120 | Chem. Sci., 2021, 12, 8115–8122
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ꢁ
transportation capillary was maintained at 320 C. For tandem R01DC010777, P50DE026787, P30CA014520, P01CA250972,
MS analysis, either collision-induce dissociation (CID) or high- T32GM008692, F30CA250263, and TL1TR002375. The MS
energy collisional dissociation (HCD) were performed. The instruments were supported by grants NIH-NCRR S10RR029531
precursor ions were isolated with a mass window width of and S10OD025084. MX acknowledges NSF Graduate Research
0.8 Da, and normalized collision energy (NCE) was set to 20– Fellowship Program (DGE-1747503). LL acknowledges a Vilas
45%. Other MS instrumental parameters were set to default Distinguished Achievement Professorship and Charles Mel-
values without any further optimization. Three replicates were bourne Johnson Professorship with funding provided by the
run to collect nanoESI MS data.
Wisconsin Alumni Research Foundation and University of
For molecular imaging of FAs, tissue section samples were Wisconsin-Madison School of Pharmacy.
analyzed using a Bruker rapieX MALDI Tissuetyper TOF mass
spectrometer (Bruker Scientic, LLC, Bremen, Germany) tted
with a Smartbeam 3D Nd:YAG (355 mm) laser. Smartbeam laser
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8122 | Chem. Sci., 2021, 12, 8115–8122
© 2021 The Author(s). Published by the Royal Society of Chemistry