Spatial Localization and Quantitation of Androgens in Mouse Testis by Mass Spectrometry Imaging

Article abstract of DOI:10.1021/acs.analchem.6b02242

Androgens are essential for male development and reproductive function. They are transported to their site of action as blood-borne endocrine hormones but can also be produced within tissues to act in intracrine and paracrine fashions. Because of this, circulating concentrations may not accurately reflect the androgenic influence within specific tissue microenvironments. Mass spectrometry imaging permits regional analysis of small molecular species directly from tissue surfaces. However, due to poor ionization and localized ion suppression, steroid hormones are difficult to detect. Here, derivatization with Girard T reagent was used to charge-tag testosterone and 5α-dihydrotestosterone allowing direct detection of these steroids in mouse testes, in both basal and maximally stimulated states, and in rat prostate. Limits of detection were ～0.1 pg for testosterone. Exemplary detection of endogenous steroids was achieved by matrix-assisted laser desorption ionization and either Fourier transform ion cyclotron resonance detection (at 150 μm spatial resolution) or quadrupole-time-of-flight detection (at 50 μm spatial resolution). Structural confirmation was achieved by collision induced fragmentation following liquid extraction surface analysis and electrospray ionization. This application broadens the scope for derivatization strategies on tissue surfaces to elucidate local endocrine signaling in health and disease.

Full text of DOI:10.1021/acs.analchem.6b02242

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Technical Note
Spatial Localization and Quantitation of Androgens
in Mouse Testis by Mass Spectrometry Imaging
Diego F Cobice, Dawn E. W. Livingstone, Colin Logan Mackay, Richard
J. A. Goodwin, Lee B. Smith, Brian R. Walker, and Ruth Andrew
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02242 • Publication Date (Web): 27 Sep 2016
Downloaded from http://pubs.acs.org on September 28, 2016
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Analytical Chemistry
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Spatial Localization and Quantitation of Androgens in Mouse
Testis by Mass Spectrometry Imaging
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Diego F Cobicea,*$, Dawn EW Livingstonea,b,$, C Logan Mackayc, Richard JA Goodwind, Lee
B Smithe, Brian R Walkera and Ruth Andrewa
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a
University/British Heart Foundation Centre for Cardiovascular Science, Queen´s Medical
Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ,
UK, b Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, 15
c
George Square, Edinburgh EH8 9XD, UK. SIRCAMS, School of Chemistry, University of
Edinburgh, Joseph Black Building, The King's Buildings, West Mains Road, Edinburgh, EH9
3JJ, UK.d AstraZeneca R&D, Cambridge, CB4 0WG, UK.e MRC Centre for Reproductive
Health; Queen´s Medical Research Institute, University of Edinburgh, 47 Little France
Crescent, Edinburgh, EH16 4TJ, UK. $ Joint first author
Corresponding Author: Professor Ruth Andrew. as above
Tel: +44 (0) 131 242 6763
Fax: +44(0) 131 242 6779
Email: ruth.andrew@ed.ac.uk
* Footnote: Subsequently Biomedical Sciences Research Institute, School of Biomedical
Sciences, University of Ulster, Coleraine campus, Cromore Road, Coleraine, Co.
Londonderry, BT52 1SA, UK.
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Abstract
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Androgens are essential for male development and reproductive function. They are
transported to their site of action as bloodꢀborne endocrine hormones, but can also be
produced within tissues to act in intracrine and paracrine fashions. Because of this, circulating
concentrations may not accurately reflect the androgenic influence within specific tissue
microenvironments. Mass spectrometry imaging permits regional analysis of small molecular
species directly from tissue surfaces. However, due to poor ionization and localized ion
suppression, steroid hormones are difficult to detect. Here derivatization with Girard T
reagent was used to chargeꢀtag testosterone and 5αꢀdihydrotestosterone allowing direct
detection of these steroids in mouse testes, in both basal and maximallyꢀstimulated states and
in rat prostate. Limits of detection were ~0.1 pg for testosterone. Exemplary detection of
endogenous steroids was achieved by matrixꢀassisted laser desorption ionization and either
Fourier transform ion cyclotron resonance detection (at 150 ꢁm spatial resolution) or
quadrupoleꢀtime of flight detection (at 50 ꢁm spatial resolution). Structural confirmation was
achieved by collision induced fragmentation following liquid extraction surface desorption
and electrospray ionization. This application broadens the scope for derivatization strategies
on tissue surfaces to elucidate local endocrine signaling in health and disease.
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Introduction
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Androgens are essential regulators of male development and adult reproductive function,
primarily synthesized within the testis. During embryonic development, testicular fetal
Leydig and Sertoli cells work in concert to produce testosterone1;2. This is converted into the
most active androgen, 5αꢀdihydrotestosterone (DHT), by 5αꢀreductases in peripheral tissues
to promote masculinization and normal size of target organs3ꢀ5. In postnatal life a second
population of (adult) Leydig cells develops superseding the fetal population; these Leydig
cells complete all steps of steroidogenesis to produce and secrete testosterone throughout
adulthood6. Additional androgens, androstenedione (A4) and dehydroepiandrosterone
(DHEA), are synthesized in the adrenal glands in many species but not rats or mice7;8.
Androgens have pleiotropic functions in the testis, e.g. completion of meiosis, differentiation
of spermatids, initiation of spermatogenesis at puberty and maintenance of this process in the
adult9. Dysregulation of these processes may lead to testicular dysfunction and infertility. In
contrast, increased androgen signaling plays a role in pathologies, including benign prostatic
hyperplasia (BPH) and prostate cancer 10;11
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Liquid chromatographyꢀtandem mass spectrometry (LCꢀMS/MS) is the method of choice for
quantifying circulating androgens, but little information is available relating circulating to
tissue levels due to analytical limitations. While androgens may be quantified in extracts of
biopsies7;12, this does not spatially align steroid levels with subꢀregions, e.g. cell types or
tumours. Retaining spatial distribution of androgens can be crucial, for examples in androgenꢀ
sensitive prostate tumours, where the cellular activities of 5αꢀreductases are critical to the
local exposure to DHT. Mass spectrometry imaging (MSI) is gaining traction as a
bioanalytical tool to map spatial distribution of small molecules in tissues and identify,
localize and relatively quantitate compounds in complex biological matrices13. Many small
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molecule applications of MSI pertain to drug discovery, but we recently validated a MSI
approach for assessing the spatial distribution of glucocorticoids in murine brain14. Onꢀtissue
chemical derivatization (OTCD) using Girard T (GirT) reagent overcame poor target
ionization efficiency, ion suppression and the increased m/z helped avoid interference of
matrixꢀrelated background in the lower mass range. GirT reagent reacts with ketones15 and
enhances sensitivity in the measurement of lipophilic steroids within tissue matrix, but the
approach requires skillful and flexible manual application. Glucocorticoids are pregnene
steroids, with several ketones, the most reactive being at C3 in the Aꢀring, conjugated with a
C4ꢀ5 double bond (Supporting Information: Nomenclature of steroids, Figure Sꢀ1). The
endogenous androgens, testosterone and androstenedione, contain a similar ketonic function
and may also be derivatized using hydrazineꢀbased reagents15 (Supporting Information:
Derivatization Reactions, Figure Sꢀ1). A recent report suggests derivatization with GirT is
useful for MSI of testosterone in tissue16. However, within tissue, testosterone levels must be
interpreted in conjunction with those of the more potent DHT. The Aꢀring of DHT is reduced
and the ketone less activated (not allylic to the ꢀ4ꢀ5 double bond) and thus less amenable to
derivatization.
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Here we describe how OTCD with MSI can spatially localize testosterone and DHT in rodent
reproductive tissues. Translational challenges and methodological improvements to reduce
diffusion and increase robustness by inclusion of automated spraying technology are reported
for the first time.
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Experimental
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Sources of Chemicals: Internal standard [2H]8ꢀcorticosteroneꢀ2,2,4,6,6,7,21,21 (d8ꢀCORT,
Cambridge Isotopes; MA, USA); unlabeled steroids (Steraloids Inc, PA, USA). Solvents were
glassꢀdistilled HPLC grade (Fisher Scientific, Loughborough, UK). Other chemicals were
from SigmaꢀAldrich (Dorset, UK) unless stated. Room temperature (RT) was 18ꢀ21oC.
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Animals and biomatrix collection: C57BL/6 mice (Harlan Olac Ltd, Bicester, UK) were
studied under UK Home Office license. To stimulate androgen biosynthesis, human chorionic
gonadotrophin (hCG, 20 IU, Organon, The Netherlands) or vehicle (saline) was administered
by subcutaneous injection to male mice (aged 8ꢀ10 weeks; n=3/group) and testes and blood
collected following cull by CO2 16 h later. Plasma (EDTA) was prepared by centrifugation
(10,000 g, 5 min, 4oC). Tissues were snap frozen in liquid nitrogen and stored (ꢀ80˚C).
Concentrations of testosterone in plasma were quantified by LCꢀMS/MS17. Prostates were
similarly collected from male Sprague Dawley rats (Harlan Olac Ltd).
Reaction efficiency: LC-MS/MS analysis of non-derivatized and derivatized steroids:
Steroid (1 ꢁg/10 ꢁL methanol) was derivatized with GirT (5 mg/mL in methanol containing
0.1% trifluroacetic acid (TFA)) by heating (40oC, 60 min) and the reaction stopped with water
(50 ꢁL). Nonꢀderivatized and derivatized steroids were analyzed by LCꢀMS/MS (QTrap 5500
triple quadrupole MS (Sciex, Warrington, UK) interfaced with an Acquity UPLC (Waters,
Manchester, UK)). Separation was achieved using a Kinetix C18 column (Phenomenex,
Macclesfield, UK; 150 cm, 3 mm, 2.6 ꢁm; 35oC). Initial mobile phase conditions were water
with 0.1% formic acid:methanol (70:30), changing to 20:80 from 1ꢀ10 minutes and then
sustained until 15.5 minutes. Selected ion monitoring detected nonꢀderivatized and
derivatized steroids respectively; testosterone (m/z 289.1, 402.3) and DHT (m/z 291.1, 404.2)
with electrospray (ESI) MS (source temperature 30oC, ion source gases (GS1, GS2 50 and 70
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respectively), CAD Low, ion spray voltage 5500 V, curtain gas 40 psi, entrance potential, 10
V).
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Imaging Instrumentation: MSI was performed firstly, by 12T SolariX MALDIꢀFTICRꢀMS
(Bruker Daltonics, MA, US) employing a Smartbeam 1 kHz laser, operated with SolariX
control v1.5.0 (build 42.8), Hystar 3.4 (build 8) and FlexImaging v3.0 (build 42).
Confirmatory onꢀtissue collision induced dissociation (CID) was carried out by Liquid
extraction surface analysis (LESA)ꢀnanoESIꢀFTICRꢀMS (Triversa Nanomate®, Advion, New
York, USA). Secondly high spatial resolution imaging was performed using a MALDI qꢀTOF
MS (MALDI Synapt G2 HDMS, Waters). Regions of interest (ROIs) were defined. Image
files were generated in Mass Lynx (v4.1), then viewed in HDI Imaging (v1.2).
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Tissue sectioning and mounting: Tissue was embedded in gelatin (50 %w/v). Topꢀdown
(horizontal) sections of testes (10 µm) were thaw mounted onto conductive indium tinꢀoxide
(ITO)ꢀcoated glass slides (Bruker Daltonics, Bremen, GmbH) and stored in a vacuum
desiccator (RT, 1 h) and then at ꢀ80˚C. Adjacent sections were stained using haematoxylin
and eosin. After fixation in cold acetone, tissue sections were examined using an optical
microscope (40X, Leica Microsystems Inc, Bannockburn, IL, USA) with CCD camera
(Hitachi, 3969, Japan).
Detection of endogenous steroids in rodent tissues without derivatization: Tissues were
prepared and matrix applied (αꢀcyanoꢀ4ꢀhydroxycinnamic acid; CHCA) as below.
Detection of endogenous steroids in rodent tissues following OTCD: From ꢀ80oC, tissue
sections were dried in a vacuum desiccator (20 mins). Derivatization reagent (GirT, 5 mg/mL
methanol: water (80:20) with 0.1% TFA containing d8ꢀCORT (10 µg/mL) was applied by
artistic airbrush14, with a reagent density of 0.11 mg/cm2. The procedure for reagent and
matrix application14 was automated for higher resolution imaging. The reagent was applied
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(flow rate 90 µL/min) by automated sprayer (HTX Technologies, Carrboro, North Carolina,
US). Nebulization gas was nitrogen (10 psi). The nozzle spray (100oC, positioned ~35 mm
from the target) was deposited (linear velocity 850 mm/min, offset spacing 3 mm). Ten passes
were performed leading to a matrix density of 0.21 mg/cm2, matched to manual spraying14.
On alternate passes, the spray pattern was offset by 1.8 mm. After derivatization, the slide
was placed in a temperature/humidity controlled chamber (Memmert HPP 110, Schwabach,
GmbH; 40oC, 80% relative humidity) in a sealed slide box. The tissue was incubated (60 min,
40°C), then allowed to cool and dry in a vacuum desiccator (RT, 15 min) to remove the
condensed water prior to matrix deposition.
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Matrix application: CHCA (10 mg/mL in acetonitrile (80%) + 0.2% v/v TFA) was applied by
a pneumatic TLC sprayer (20 mL/slide, nitrogen flow 7.5 L/min, distance 20 cm from target).
Each manual pass took ~1 s and was repeated with 5ꢀ10 s between passes, until uniform
coating was achieved. The tissue section was allowed to dry (RT) and stored in a desiccator.
For higher resolution studies, the matrix was applied by automated sprayer as above except
that the nozzle was 90oC and linear velocity 1100 mm/min.
MALDI-FTICR-MSI analysis: Optical images were scanned (Canon LiDEꢀ20, Canon, UK).
MSI analysis was performed using constant accumulation of selected ions (CASITM) using a
80 Da isolation window centered at 435 Da yielding a 2 Mword timeꢀdomain transient. Laser
spot diameter was 100 µm and raster spacing 100–300 µm. Laser shots were 800 and power
optimized for consistent ion production. MSI data were subject to window normalization to
m/z of 468.3064 (GirTꢀd8ꢀCORT). Mass precision was typically ±0.0005 Da. Average
abundances was assigned from the summed spectra within ROIs. Neutral testosterone and
DHT were analyzed (without derivatization) in positive mode at m/z 289.2098 and m/z
291.2112. Ions of derivatives were monitored in positive mode; m/z 402.3115 (GirTꢀ
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testosterone) and 404.3271 (GirTꢀDHT). The CHCA matrix ion at +ve m/z 417.0483, along
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with GirTꢀd8ꢀCORT, were monitored to assess uniformity of matrix application.
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Higher Spatial Resolution Analysis: Using a MALDI qꢀTOF MS, the ROI was defined and
the spatial resolution set (50 ꢁm). Positive ion data were acquired in sensitivity mode (target
enhancement at m/z 402) with 350 laser shots/raster position using a 1 kHz laser.
Optimization was achieved by tuning acquisition settings while collecting data from a control
spot of analytes (0.5 ꢁL, 0.1 g/mL of standard at ~2 ꢁm manually spotted with equal volume
of derivatization solution/matrix). Ions formed by derivatives were monitored in positive
mode; m/z 402.31 (GirTꢀtestosterone), m/z 404.33 (GirTꢀDHT). Mass filter windows were
selected with a precision of ±0.04 Da. Data were normalized by total ion current.
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Liquid extraction surface analysis (LESA)-ESI-FTICR-MS: Steroids within tissue sections
were derivatized14 and analyzed immediately using LESAꢀnanoESIꢀFTICRꢀMS with the 12T
SolariX ESIꢀMALDI source; solvent (methanol: water, 50:50 with 0.1% v/v of formic acid),
pickꢀup volume: 1.5 µL, dispense volume, 1.2 µL at 0.2 mm from surface, droplet rest time
(delay) 5 s and aspiration volume of 1.4 µL at 0.0 mm from surface. Ions were detected
between m/z 250ꢀ1500, with an isolation window of 0.1 Da, yielding a 2 Mword timeꢀdomain
transient. Ions of GirTꢀhydrazones (as MALDI) were isolated (30 s) prior to selection of m/z
400.3±5 for CID performed at 32 eV.
Limits of Detection were assessed by spotting serial dilutions of steroid solutions (0.1–100
pg) onto slides and also control sections (murine brain) and assessed by MALDIꢀFTICRꢀMS.
Data were compared using Student’s tꢀtest (Statistica® version 8.0, StatSoft, Inc., Tulsa, OK,
USA).
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Results and Discussion
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Mass Spectral Characterization of Steroids: The mass spectra of nonꢀderivatized androgen
standards contained protonated molecular ions (testosterone m/z 289.2098; DHT m/z
291.2112). Initial imaging attempts to detect nonꢀderivatized androgens in mouse testes were
unsuccessful, as signal to noise ratios (SNR) <2 (Figures 1b, 1g), even following hCG
stimulation. This resembles glucocorticoids, which are also undetectable in their native state
by ourselves14 and others16. Accordingly, GirT derivatives were formed and reactions
monitored using ESI, which allowed concomitant assessment of native and derivatized forms.
With ESI, the steroid signal intensities were boosted by ~20ꢀ30x by derivatization. However,
reaction efficiencies differed with DHT, reacting ~5 times more slowly than testosterone, due
to its less reactive 3ꢀketone.
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Reaction conditions were subsequently optimized onꢀtissue using testosterone. The MALDI
MS spectra of the GirTꢀhydrazones yielded [M]+ ions, with masses at m/z 402.3114 for
testosterone (Figure 1k) and m/z 404.3264 for DHT (Figure 1l), in close agreement with
theoretical masses. As before, GirT derivatives yielded spectra dominated by the molecular
ion and high resolution MS overcame the challenge of selecting specific analytes from high
abundance ions in the low mass range emanating from matrix and tissue. Structural
confirmation was performed within tissue by CID using LESAꢀESIꢀFTICRꢀMS, allowing
isolation experiments to be performed with greater sensitivity than could be achieved during
MALDI imaging (Supporting Information: CID mass spectra, Figure Sꢀ2). CID of GirTꢀ
testosterone generated fragment ions (m/z 343 and 315) characteristic of loss of the quaternary
amine tag [Mꢀ59]+ and carbon monoxide [Mꢀ87]+ respectively from the derivatized group.
Similar fragmentation occurred with GirTꢀDHT. Proposed patterns (Supporting Information:
Proposed Fragmentation Patterns, Figures Sꢀ2) agreed with the fragmentations of GirT
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derivatives of glucocorticoids14 and also those of GirP steroid hydrazones, which may form
stable five membered rings18. Further androgens (androstenedione, DHEA) were also
successfully derivatized with GirT (Supporting Information: Mass spectrum, Figure Sꢀ3).
Notably GirTꢀDHEA gave the same precursor ion as testosterone and fragmented similarly
under LESA conditions, unless high energy was applied.
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OTCD and matrix application were adapted for automated spraying, allowing greater control
of crystal size and topological homogeneity, aiming to improve spatial resolution and reduce
the likelihood of hot spots. Previously deposition of methanolic solutions was by either
manual TLC sprayer or airꢀbrushes, where distance from target and nozzleꢀnozzle
reproducibility were difficult to control, varying between suppliers and even lots. Nonetheless
a good spray mist could be achieved leading to a homogenous layer of reagent, good analyte
extraction and desirable coꢀcrystallization. However, reproducibility was highly analyst
dependent and analyte diffusion was a concern through repeated wetting.
Sublimation (solventꢀfree) was piloted, leading to smaller crystals size but poor tissueꢀ
extraction, particularly with hydrophobic analytes. Shimma et al report a successful dual
approach of sublimation and airbrush to reduce crystal size16. Ultimately automated
deposition was used, improving reproducibility and crystal size homogeneity but requiring
extensive optimization of spray parameters (nozzle temperature, nitrogen flow, solvent
composition, solvent flow rate, surface tension) to achieve good analyte extraction. A balance
between “not too wet”, promoting diffusion, and “not too dry”, impeding analyte extraction,
was struck. Our experience is that, while reproducibility is improved by automated spraying,
better sensitivity/extraction may still be observed using airꢀbrush. Furthermore, automatic
sprayers require instrumentꢀspecific optimization.
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Relative quantitation of derivatized steroids: CASI was used to maximize signal intensity
and limits of detection (LODs) of derivatized testosterone were ~ 0.1pg (offꢀtissue) and 1pg
(onꢀtissue; SNR 21). GirT derivatives of testosterone yielded ions of similar intensity to GirTꢀ
d8ꢀCORT; similar ionization efficiency is anticipated14, for steroids with an activated Aꢀring
ketone. However, the yield of GirTꢀDHT was lower due to reduced reactivity of the nonꢀ
conjugated ketone moiety. Therefore relative quantitation between testosterone and DHT
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requires normalization, readily achievable with stableꢀisotope labeled internal standards. C
labelling is preferred to deuterium due to the potential for loss of deuterium during
derivatization or fragmentation19.
Detection of endogenous steroids in rodent tissues
GirT derivatives of androgens were successfully detected using two imaging MS platforms.
Initially studies were performed by MSI (with FTICR) at 150 µm spatial resolution in murine
testes, detecting steroids with mass accuracy of ±5 ppm from their theoretical monoꢀisotopic
mass (Figure 1k, 1l). Androgens were detected in control testes (Figures 1c, 1h) with SNR
of 22±5 and 43.5±4 for derivatized testosterone and DHT respectively. Testosterone and DHT
were in higher abundance following stimulation with hCG (Figures 1d, 1i); SNR 265±11 and
387±13 respectively. The absolute signal intensity of testosterone and DHT relative to
d8CORT were increased ~2.5 and ~1.8 fold by hCG (Figure 2e, 2j) respectively.
Corresponding concentrations of testosterone in plasma rose from 4.5±0.7 to 29.6±1.8 nM
following hCG, typical of the protocol20. Shimma et al reported an increase of greater
magnitude, but over a shorter timecourse16.
The distributions of testosterone and DHT following hCG stimulation were assessed at higher
spatial resolution (50 µm) using a MALDIꢀqꢀTOF instrument. In CASI mode on the SolariX
(800 laser shots/pixel) a decline in sensitivity after 20000 pixels was observed, possibly due
to an accumulation of matrix/reagent ion clusters in the source; this was not observed by
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qTOFꢀMS. The steroid derivatives yielded the same molecular ions as with FTICR. The mass
resolution was ~20000 (at m/z 400) as opposed to 350000 and the difference in mass accuracy
of selected ions was <5 ppm versus their theoretical monoisotopic masses (Figure 2g).
Potential interfering ions within the mass window were not evident in either the qTOF or the
FTICR spectra from mouse testes. DHT was also detected, but with a different spatial
distribution. Testosterone was mainly localized within the seminiferous tubules (Figure 2a,
2d), whilst DHT was mainly observed in the interstitium/Leydig cells (Figure 2b, 2e).
Although Shimma et al16 report Leydig cell localization of testosterone, their images display a
clear signal in seminiferous tubules, in keeping with ours.
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The method was subsequently applied to prostate tissue. 5αꢀReductases are pharmacological
targets within prostate, since suppression of DHT production attenuates growth of both
hyperplastic and cancerous tissue. Both testosterone and DHT could be detected SNR>10
(Figures 1n,1o,1p), suggesting that the MSI approach may open doors to pathological
investigations of cellꢀspecific androgen synthesis in prostate disease.
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Derivatization permits detection of poorly ionizable endogenous androgens in target tissues
by MSI. The use of Gir T reagent affords low limits of detection, similar to glucocorticoids14.
OTCD proceeded rapidly for testosterone but was less efficient for DHT. However both were
detected in tissue. For relative quantitation, commercial stableꢀisotope labeled androgens
would allow data collection within a narrower mass window of ± 5 or 10 Da, (as opposed to
80 Da using d8CORT), potentially improving sensitivity.
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OTCD coupled with MALDIꢀFTICRꢀMSI successfully detected androgens in murine testes.
A proofꢀofꢀprinciple experiment, representative of healthy biological variations in tissue
steroid levels, was performed using hCG to maximally stimulate androgen synthesis,
achieving an increase in plasma testosterone which was also reflected in testicular steroids.
Initial studies were performed at 150 µm resolution, detecting androgens in testes but without
meaningful molecular histology. The seminiferous tubules of mice are ~300 ꢁm in diameter
and hence smaller laser bores were required to reveal the characteristic tissue structure,
although this may be less challenging in larger species. Higher resolution imaging (50μm),
localized testosterone and DHT to different compartments of the adult mouse testis.
The need for higher resolution imaging brings with it concerns over analyte diffusion, matrix
crystal size and tissue integrity. Manual application of matrix and reagent, while effective,
was subject to variability between operators. The automated application allows better tissueꢀ
tissue reproducibility and improved homogeneity of matrix coverage compared with TLC
sprayers or airbrushes. Future optimization of OTCD and matrix application by sublimation
may allow even higher spatial resolution, possibly 5 µm approaching cellular resolution, now
obtainable with new MALDI systems.
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MSI with OTCD is a powerful tool to study the regional variation in abundance of androgens
in tissues and here we broaden scope of the technique to reproductive biology. In translating
to nonꢀrodent tissues, the presence of isobaric steroids, highly abundant DHEA, as well as
epiꢀtestosterone, must be considered. Although underivatized DHEA fragments differently
from testosterone under tandem MS conditions, the predominant fragment of the derivatives
within FTICR or the qTOF is the derivatizing group, militating against their discrimination in
the absence of chromatography. Other approaches such as MSn as reported with the
IMScope®16 or ion mobility may permit isomeric separation, as required by translational
research.
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We acknowledge funding from the Medical Research Council (MC_PC_12014), the British
Heart Foundation (RGꢀ05ꢀ008) and its Centre for Research Excellence (BHF RE/08/001).
BRW is a Wellcome Trust Senior Investigator.
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Conflict of Interest Disclosure
No conflicts of interest to declare.
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Figure captions
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Figure 1
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Molecular imaging by MALDIꢀFTICRꢀMSI of androgens analyzed intact and as Girard
T derivatives in murine testes. Nonꢀderivatized steroids could not be detected testes even
following stimulation with human chorionic gonadotrophin (hCG). However, upon
derivatization, testosterone and 5αꢀdihydrotestosterone (DHT) were detected. Optical images
of a cryosection of murine testis (a, f) or rat prostate (m). Molecular image of: (b) nonꢀ
derivatized testosterone at m/z 289.2098±0.005Da in an hCGꢀstimulated mouse. Derivatized
testosterone at m/z 402.3114±0.0005Da in testes from (c) control and (d) hCG stimulated
mouse. (e) The relative abundance of testosterone (corrected for internal standard, d8ꢀ
corticosterone (d8ꢀCORT)) was increased ~2.5 fold following hCG stimulation. Molecular
image of: (g) nonꢀderivatized DHT at m/z 291.2112±0.0005Da in an hCG stimulated mouse.
Derivatized DHT at m/z 404.3264±0.0005Da in testes from (h) control and (i) hCG stimulated
mouse. (j) The relative abundance of DHT (corrected for internal standard) was increased
~1.8 fold following hCG stimulation. Derivatised testeosterone (n) and DHT (o) in rat
prostate. Representative FTICRꢀMS spectrum of (k) testosterone and (l) DHT hydrazone in
mouse testes and (o) both steroidal derivatives in rat prostate showing excellent agreement
(mass accuracy ±5 ppm) with simulated theoretical isotopic distribution pattern (embedded).
Data are mean±SEM; n=3 mice per group. cps = counts per second. Scale bar (2 mm). Signal
intensity is depicted by color on the scale shown. ** = p<0.01 compared by Student tꢀtest.
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Figure 2
Molecular imaging of testosterone and 5αꢀdihydrotestostrone (DHT) Girard T
derivatives in rodent testes at 50 µm spatial resolution by MALDIꢀqTOFꢀMSI. Images of
testes from mice stimulated with human chorionic gonadotrophin (a) Derivatized testosterone
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at m/z 402.31±0.02Da. (c) 15X zoom image of (a) (b) Derivatized DHT at m/z
404.33±0.02Da (d) 15X zoom image of (b). (e) 15X zoom of haematoxylin and eosin stained
section of mouse testis. (f) cartoon of testicular architecture (ST = seminiferous tubule, LC =
Leydig cells. (g) (j). Mass Spectra of steroid derivatives detected in testes detected by
MALDIꢀqTOFꢀMSI. Signal intensity is depicted by color on the scale shown. Scale bar
(2mm).
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Supporting Information
Figure Sꢀ1: Steroid Nomenclature and Derivatization Reaction Schemes to form Girard T
derivatives of common endogenous androgens.
Figure Sꢀ2: Mass spectra and proposed fragmentation patterns of Girard Tꢀderivative of
testosterone and dihydrotestosterone collected following liquid extraction surface analysis
with nanoESIꢀFTICR collision induced dissociation.
Figure Sꢀ3: MALDIꢀFTICRꢀMS spectra and collisionꢀinduced fragmentation of a mixture of
androstenedione and dehydroepiandrosterone derivatized with Girard T reagent.
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Reference List
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Masumori, N.; Ichikawa, T.; Mizokami, A.; Sugimura, Y.; Nonomura, N.; Sakai, H.;
Honma, S.; Harada, M.; Kubota, Y., BMC Cancer 2014, 14, 717.
13. Heeren, R.M.; Chughtai, K., Chem Rev 2010, 110, 3237ꢀ3277.
14. Cobice, D.F.; MacKay, C.L.; Goodwin, R.; McBride, A.; LangridgeꢀSmith, P.; Webster,
S.P.; Walker, B.R.; Andrew, R., Analytical Chemistry 2013, 85, 11576ꢀ11584.
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16. Shimma, S.; Kumada, H.ꢀO.; Taniguchi, H.; Konno, A.; Furuta, K.; Matsuda, T.; Ito, S.,
Analytical Bioanalytical Chemistry 2016, DOI 10.1007/S00216ꢀ016ꢀ9594ꢀ9.
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N.Z.M.; Andrew R, Talanta 2015, 131, 728ꢀ735.
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Endocrinology 2016, 157, 2479ꢀ2488.
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Molecular imaging by MALDIꢀFTICRꢀMSI of androgens analyzed intact and as Girard T derivatives in murine
testes. Nonꢀderivatized steroids could not be detected testes even following stimulation with human
chorionic gonadotrophin (hCG). However, upon derivatization, testosterone and 5αꢀdihydrotestosterone
(DHT) were detected. Optical images of a cryosection of murine testis (a, f) or rat prostate (m). Molecular
image of: (b) nonꢀderivatized testosterone at m/z 289.2098±0.005Da in an hCGꢀstimulated mouse.
Derivatized testosterone at m/z 402.3114±0.0005Da in testes from (c) control and (d) hCG stimulated
mouse. (e) The relative abundance of testosterone (corrected for internal standard, d8ꢀcorticosterone (d8ꢀ
CORT)) was increased ~2.5 fold following hCG stimulation. Molecular image of: (g) nonꢀderivatized DHT at
m/z 291.2112±0.0005Da in an hCG stimulated mouse. Derivatized DHT at m/z 404.3264±0.0005Da in
testes from (h) control and (i) hCG stimulated mouse. (j) The relative abundance of DHT (corrected for
internal standard) was increased ~1.8 fold following hCG stimulation. Derivatised testeosterone (n) and DHT
(o) in rat prostate. Representative FTICRꢀMS spectrum of (k) testosterone and (l) DHT hydrazone in mouse
testes and (o) both steroidal derivatives in rat prostate showing excellent agreement (mass accuracy ±5
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ppm) with simulated theoretical isotopic distribution pattern (embedded). Data are mean±SEM; n=3 mice
per group. cps = counts per second. Scale bar (2 mm). Signal intensity is depicted by color on the scale
shown. ** = p<0.01 compared by Student tꢀtest.
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Molecular imaging of testosterone and 5αꢀdihydrotestostrone (DHT) Girard T derivatives in rodent testes at
50 µm spatial resolution by MALDIꢀqTOFꢀMSI. Images of testes from mice stimulated with human chorionic
gonadotrophin (a) Derivatized testosterone at m/z 402.31±0.02Da. (c) 15X zoom image of (a) (b)
Derivatized DHT at m/z 404.33±0.02Da (d) 15X zoom image of (b). (e) 15X zoom of haematoxylin and
eosin stained section of mouse testis. (f) cartoon of testicular architecture (ST = seminiferous tubule, LC =
Leydig cells. (g) (j). Mass Spectra of steroid derivatives detected in testes detected by MALDIꢀqTOFꢀMSI.
Signal intensity is depicted by color on the scale shown. Scale bar (2mm).
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