An Electron Acceptive Mass Tag for Mass Spectrometric Imaging

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An Electron Acceptive Mass Tag for Mass Spectrometric ImagingGuided Synergistic Targeting to Mice Brain Glutamate Receptors Ruowei Jiang, Juan Zhang, Si Zou, Shanshan Jia, Xiebin Leng, Yinghua Qi, Xuekun Zou, Baojie Shen, Weidan Li, Wenting Lu, and Hongying Zhong ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00580 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on December 24, 2018

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ACS Chemical Neuroscience

An Electron Acceptive Mass Tag for Mass Spectrometric Imaging-Guided Synergistic Targeting to Mice Brain Glutamate Receptors

Ruowei Jiang#, Juan Zhang#, Si Zou#, Shanshan Jia, Xiebin Leng, Yinghua Qi, Xuekun Zou, Baojie Shen, Weidan Li, Wenting Lu, Hongying Zhong*

Mass Spectrometry Center for Structural Identification of Biological Molecules and Precision Medicine Institute of Public Health and Molecular Medicine Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, Hubei 430079, P. R. China

#

These authors contribute equally to this work

*

To

whom

correspondence

should

be

[email protected]

1

ACS Paragon Plus Environment

addressed.

Email:

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract Dysfunctional glutamate receptors (GluRs) have been implicated in neurological disorders and injuries. splicing

variants

Hetero-tetrameric assemblies of different GluR subunits or

have

pharmacological properties.

distinct

spatiotemporal

expression

patterns

and

Mass spectrometric imaging of GluRs-targeted small

molecules is important for determining the regional preferences of these compounds. We report herein the development of a mass tag covalently bonded with glutamate or N-methyl-D-aspartate that functions as both an electron acceptor to generate mass spectrometric signals on irradiated (Bi2O3)0.07(CoO)0.03(ZnO)0.9 nanoparticles with the 3rd harmonic (355 nm) of Nd3+:YAG laser and as the core component to target bilobed clamshell-like structures of GluRs. same tag ion.

In this approach, different molecules produce the

It provides a new avenue for quantitative assessment of spatial

densities of different compounds, which cannot be achieved with well-established stable isotope labeling technique due to different ionization efficiency of different compounds.

Various co-existed endogenous molecules are also simultaneously

detected for investigation of overall physiological changes induced by these compounds.

Because semiconductors do not generate background peaks, this

method eliminates interferences from organic matrix materials that are used in regular MALDI (Matrix Assisted Laser Desorption Ionization).

The localized ionization

provides high spatial resolution that can be down to sub-micrometers.

Keywords: Mass Spectrometric Imaging; Electron Acceptive Tag; Glutamate Receptors; Photoelectron Capture Ionization; Semiconductor Nanoparticles

2


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Introduction Glutamate receptors (GluRs) refer to a large family of synaptic proteins expressed on membranes of neuronal cells in central nervous system (CNS).

The major excitatory

neurotransmitter glutamate in CNS is sensed by either ionotropic glutamate receptors (iGluRs) which are ligand-gated ion channels，or metabotropic glutamate receptors (mGluRs) which are G protein-coupled receptors.

Through binding with glutamate

(Glu), glycine (Gly), N-methyl-D-aspartate (NMDA) or other ligands, GluRs mediate excitatory neurotransmission crucial for basic brain development and functions such as synaptic plasticity, memory formation and learning.1-4

However, overactive

GluRs cause neurodegeneration and neuronal damage that are related with numerous pathological conditions ranging from Alzheimer’s disease to addiction.5-7

Decades

of research efforts have been focused on developing pharmaceutical compounds that can target GluRs and prevent neurons from excitotoxicity.

The pharmacological challenge in designing therapeutic compounds results from the structural and functional diversities of GluRs.

For example, iGluRs are generally

assembled as hetero-tetramers and differ in subunit compositions that dynamically change with different developmental stages and brain locations.

The structural

diversities of GluRs ascribes to various encoding gene families and alternative splicing.8-10

Pre-synaptically released glutamate can bind to different subtypes of

GluRs and provokes different responses.11-16

The dynamic combination of subunit

assembles with strikingly different spatiotemporal expression patterns results in the functional heterogeneity and multiplicity of receptor subtypes in the CNS.17 Although the detailed molecular mechanisms remain unclear, it has been proposed that these dynamic changes have important physiopathological roles.18-20

For

example, differential expression of NMDA receptors (NMDARs) within synaptic and non-synaptic sites of hippocampal neurons have been found in the development of Alzheimer’s disease.21

Heteromerization within different subunits not only confers the functional diversities 3



Page 4 of 32

of iGluRs and mGluRs but also brings about the versatile binding affinities with different

ligands

that

are

pharmacologically

interesting.22,23

Numerous

pharmacological tools have been developed to target GluRs through different mechanisms24-27.

Correlating individual synaptic signaling induced by these

pharmacological molecules requires a quantitative visualization of the overall spatial distribution of GluRs-targeted compounds.

Electron microscopy28-30 and

fluorescence microscopy31-33 have ever been the primary imaging tools by which known or predicted molecules are detected with immunogold or fluorescence labeling. These techniques provide very high spatial resolution.

Among those techniques,

ensemble imaging methods such as stimulated emission depletion (STED)34-36 and saturated

structure-illumination

sub-diffraction-limit resolution.

microscopy

(SSIM)37-39

have

achieved

And the detection limit of stochastic optical

reconstruction microscopy (STORM)40-42 and photoactivation localization microscopy (PALM)43-45 are even down to the level of single molecules.

Mass spectrometry imaging (MSI) has emerged as a new technique for deep unraveling of the spatial distribution of various molecules present in tissues.

With

the MSI technique, not only predicted molecules are localized but also other co-existed molecules can be simultaneously imaged through full scans of ions. Currently, although there is no way to visualize intact glutamate receptor proteins due to difficulties in ionization, images of spatial distributions of competed exogenous and endogenous ligands provide experimental evidences for understanding the concerted action of synaptic machines.

It provides a unique way to investigate overall

physiological changes induced by pharmacological molecules.

Several mass

spectrometric imaging methods have been established including desorption electrospray ionization (DESI),46-50 matrix assisted laser desorption ionization (MALDI), 51-53 secondary ion mass spectrometry (SIMS)54-56 and so on.

Because of

the convenient availability, MALDI imaging has been widely used for in situ analysis of biological molecules by scanning a finely tuned laser beam across the surface of matrix-coated tissue sections.

Our recently developed laser activated electron 4


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tunneling (LAET) imaging57-60 eliminates the use of organic matrix materials, and associated background interferences as well as uneven distribution of matrix crystals. In this approach, tissue slices are simply put on surfaces of thin films of (Bi2O3)0.07(CoO)0.03(ZnO)0.9 nanoparticles irradiated with the third harmonic (355 nm) of a Nd3+:YAG laser, which is brought to about 15 μm focus.

The localized

photoelectron capture ionization provides enough spatial resolution for the investigation of brain tissue slices.

Heating effects are decreased because it is not

enough for the excitation of photoelectrons. The remaining common challenge is the quantitative analysis of different molecules.

Because different molecules have

different ionization efficiency, development of otherwise new techniques are demanded to supplement stable isotope labeling.

In this work, an electron acceptive tag (EAT）has been invented for LAET imaging of mice brain slices.

Glu, NMDA and their tert-butyl esterified counterparts were

covalently bonded with the fluorenyl group of FMOC-Cl (9-fluorenylmethyl chloroformate) through a flexible linker.

Because of high electron affinity, the

common fluorenyl group in different compounds exothermically captures low-energy photoelectrons.

Then electron-directed specific fragmentations generate the same

negative fluorenyl ion from different compounds for quantitative analysis.

While the

fluorenyl group is designed to generate mass spectrometric signals, Glu and NMDA parts are expected to maintain the binding with receptor proteins.

It has been

demonstrated that the electron acceptive mass tag not only generates mass spectrometric signals but also prevents the closure of bilobed clamshell-like structures of targeted receptor proteins.

Results and Discussion Design of the electron acceptive tag.

As we know, absolute mass spectrometric

intensities of different compounds are not correlated well with their quantities due to different ionization efficiencies.

In order to evaluate the densities of Glu, NMDA as

well as different derivatives in different regions of brains, we designed an electron 5



Page 6 of 32

acceptive mass tag so that different compounds can generate the same fragment ion for quantitative analysis.

The design philosophy was shown in Figure 1 (A), in

which V0, V1, V2 and V3 represent voltages applied to the sample plat, extraction plate, hexapole and aperture, respectively.

Use of photo-reactive semiconductor

nanoparticles instead of organic matrix materials for ionization and dissociation of neutral molecules is one of major innovations in EAT-LAET approach. Semiconductor (Bi2O3)0.07(CoO)0.03(ZnO)0.9 nanoparticles co-doped with Bi3+and Co2+ were used to make films because of its better mechanical and photoelectric properties. The internal electric field present in such crystallines enables the effective separation of photo-induced electron-hole pairs for ionization and dissociation of adsorbed neutral molecules.

The fluorenyl group is the electron acceptive tag (red ball) we have designed to covalently bond with Glu or NMDA (green oval).

There are the three types of

ligand-gated ion channel glutamate receptors that are obligatory hetero-tetramers composed of splicing variants of GluN1 (1-4 a or b) and GluN2 (A-D) or GluN3 (A-B).

GluN1/GluN2 heterodimer was chosen as a model, of which GluN1 binds

with glycine and GluN2A binds with either Glu or NMDA ligands.

The large

globular clamshell-like structure with two discontinuous segments (S1 and S2) is the ligand-binding domain.

Concurrent binding of Glu and Gly is a conserved gating

principle in iGluR families which induces the closure of the clamshell-like segments and activates GluRs.

Figure 1 (B) shows that the exothermic capture of low-energy

photoelectrons occurs on the most charge deficient atom of the fluorenyl group of taged compounds indicated with an arrow (Supplementary Figure 1 and 2).

DFT

calculation of changes in free energies and enthalpies also indicates that capture of such an electron is an exothermal process and can proceed spontaneously. radical anions carrying with unpaired electrons are highly active.

Resulting

The specific

cleavage at α-positioned C-C bond next to the radical center causes the formation of the negative fluorenyl ion at m/z 165.0702.

Other α-positioned bond cleavages

including intra ring C-C bonds or C-H bond are not preferred because of higher bond 6


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energies.

The stability and detectability of this ion ascribe to the delocalization of

the acquired negative charge over the whole conjugated П system.

Although carbon

atoms of carbonyl groups are also charge deficient, the acquired negative charges cannot be well well-delocalized and stabilized.

Similarly, tert-butyl esterified

FMOC-Glu and FMOC-NMDA can generate the same negative fluorenyl ion at m/z 165.0702 through the same mechanism too.

Quantitative evaluation and detection limit.

The electron acceptive mass tag

makes it possible to quantitatively analyze different glutamate derivatives in mass spectrometric imaging.

Because different molecules have been converted into the

same gaseous fragment ion, the bias caused by different evaporation and ionization efficiencies of different compounds is eliminated (Supplementary Figure 3). Supplementary Figure 4 (A) shows the mass spectra of tert-butyl esterified derivatives of glutamate that are generated with the proposed EAT-LAET approach.

Both of

them generate the negative fluorenyl ion at m/z 165.0702 Da (error: 0.0001 Da and 0.0009 Da for FMOC-Glu(OtBu)-OH and FMOC-NMDA(OtBu)-OH respectively). In order to experimentally evaluate if different molecules can generate equal signals, 0.1 µL of a series of sample solutions containing different amounts of tagged compounds ranging from 1.25 nmol to 20 nmol has been spotted on the surfaces of thin films made of semiconductor nanoparticles.

For each sample spot, integrated

intensity of the ion was used for quantitative examination.

As shown in

Supplementary Figure 4 (B), the same amounts of different compounds FMOC-Glu(OtBu)-OH

and

FMOC-NMDA(OtBu)-OH

produce

equal

mass

spectrometric intensities.

In regular MALDI imaging, molecules are usually ionized through ubiquitous protonation processes and detected in the positive ion mode.

Compounds with

stronger proton affinities and higher concentration usually suppress the detection of those with weaker proton affinities and lower abundance.

Matrix materials and

co-existing molecules of complex biological samples in a wide concentration range 7



Page 8 of 32

are main sources of ion suppression in each single laser shot.

In contrast, the

EAT-LAET strategy is based on the detection of negative ions that are generated through photoelectron capture ionization.

Different molecules are ionized through

an electron acceptive tag that enhances the mass spectrometric signals of low abundance ions and reduces the suppression of high abundance ions.

Supplementary

Figure 5 (A) shows that the detection limit of EAT-LAET can be down to about 0.07 fmol/pixel while signal-to-noise ratio is more than 10.

It was also found in

Supplementary Figure 5 (B) that the mass spectrometric intensities of the fluorenyl ion are basically linearly correlated with the quantities ranging from 1 pmol (~0.07 fmol/pixel) to 20 nmol (~1.4 pmol/pixel).

These experimental results demonstrate

that the EAT-LAET approach is basically quantitative and sensitive.

Theoretical evaluation and experimental validation of binding affinities. Binding of tagged compounds with iGluRs or mGluRs and the prevention of the closure of bilobed clamshell-like segments have first been theoretically examined. Because iGluN2A is most abundantly expressed in almost every CNS area and its crystallographic data is available, this protein was chosen as a representative for computational modeling based on the crystal structure of iGluN2A in complex with PPDA (PDB code: 4NF6).

Supplementary Figure 6 (A) shows the comparison

between the crystal structure of iGluN2A in complex with Glu alone (PDB code: 2A5S) and simulated docking structure of iGluN2A in complex with FMOC-Glu and FMOC-Glu(OtBu)-OH

with

the

lowest

interaction

energies,

respectively.

Supplementary Figure 6 (B) represents the stereoview of the binding sites located at the cleft.

In Supplementary Figure 6 (C), it was clearly shown that the fluorenyl

derivatization of Glu and tert-butyl esterification of carboxyl groups do not change the predicted binding location.

The fluorenyl group was localized in the clamshell like

cleft by π-alkyl interaction with the nearby Val 218, Leu 14 and Lys 87.

The

derivatized or non-derivatized carboxyl group can all be stabilized by Tyr 214 through π-alkyl

interaction.

In

order

to

further

examine

if

FMOC-Glu

and

FMOC-Glu(OtBu)-OH molecules can prevent the closure of bilobed clamshell-like 8


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segments, we have computed the cleft width of iGluN2A in complex with these two compounds in comparison with that of Glu alone.

Supplementary Figure 7 (A)-(C)

show the cleft widths of iGluN2A in complex with Glu, FMOC-Glu and FMOC-Glu(OtBu)-OH respectively.

Increased distances between the two nitrogen

atoms of Gly 86 and Arg 195 in the clamshell-like structure have been observed when iGluN2A is in complex with the FMOC-Glu or FMOC-Glu(OtBu)-OH.

It was

implicated that those compounds have the capability to prevent the closure of bilobed clamshell-like segments of iGluN2A because of the steric hindrance of the bulk fluorenyl group.

Similarly, the binding conformations of NMDA, FMOC-NMDA

and FMOC-NMDA(OtBu)-OH are shown in Supplementary Figure 8 (A)-(C), respectively.

The clamshell like cleft can accommodate the electron acceptive tag

without apparent effects on the binding with glutamate and NMDA parts of tagged compounds. iGluN2A.

However, tert-butyl esterification weakens the interaction with In the case of NMDA, Glu, FMOC-Glu, FMOC-Glu(OtBu)-OH and

FMOC-NMDA, the negatively charged carboxyl groups are all stabilized by positively charged Arg 121 of iGluN2A.

But this strong charge attraction is replaced

by weak carbon hydrogen bond with Gly 172 and conventional hydrogen bond with Ser 173 in FMOC-NMDA(OtBu)-OH molecule.

The binding affinities of those

compounds towards iGluN2B, mGluR1 and mGluR7 were computationally evaluated in Supplementary Figure 9-20.

All computational results indicate that covalent

bonding of fluorenyl group with Glu, NMDA and tert-butyl esterified derivatives does not cause losses of binding affinities with iGluN2A, iGluN2B, mGluR1 and mGluR7. It also prevents the closure of bilobed clamshell-like segments.

Binding affinities were then experimentally validated with fluorescence spectroscopy. After endogenous glutamate was washed away from membrane pellets, affinities of FMOC tagged Glu, NMDA as well as tert-butyl esterified derivatives towards bulk membrane receptors were determined as shown in Supplementary Notes 3. Measured dissociation constants Kd of FMOC-Glu, FMOC-Glu(OtBu)-OH, FMOC-NMDA and FMOC-NMDA(OtBu)-OH are 0.18 μM, 1.50 μM, 0.21 μM and 9



1.12 μM, respectively.

Page 10 of 32

FMOC-Glu has the strongest binding affinity among the four

compounds towards bulk membrane proteins.

It is noted that tert-butyl esterification

does cause decreases in binding affinities of those compounds.

Binding affinities were even further validated with different neuronal cells.

As we

know, neuronal cells of cortex and hippocampus possess high affinity binding sites for Glu.

Supplementary Notes 4 show those neuronal cells treated with FMOC-Glu,

FMOC-Glu(OtBu)-OH and FMOC-NMDA(OtBu)-OH and FMOC-NMDA.

Among

them, FMOC-Glu shows the highest intensity on both cortex and hippocampal neuronal cells. cell death.

Although FMOC-NMDA also has high binding affinity, it causes

Tert-butyl esterification of FMOC-NMDA greatly decreases the binding

to both cortex and hippocampal neuronal cells, which is in accordance with the theoretical modeling. effects.

However, tert-butyl esterified FMOC-Glu brings different

While tert-butyl esterification of FMOC-Glu does not significantly change

the affinity to hippocampal neuronal cells, it does greatly changes the binding to cortex neuronal cells.

The maximum intensity of the ion at m/z 165 of

FMOC-Glu(OtBu)-OH treated cortex neuronal cells is 2.7 times lower than that of FMOC-Glu treated cells.

These experimental results implicate the heterogeneous

composition of receptor subunits with different binding affinities that changes with brain locations.

Blank mass spectrometric imaging of mice brain tissue slices.

Mice of either

two-week-old or six-week-old that were not treated with any compounds were used for blank experiments.

Figure 2 (A) shows a representative mass spectrum and how

the brain was sliced from dorsal side to ventral side.

Because long chain fatty acids

indicated with stars have specific location in the brain,57-60 we display them as site references for the localization of other species.

The spatial distribution of long chain

fatty acids C18:1 (m/z 281.2485, error: 0.0004) and C20:1(m/z 309.2793, error: 0.0008) as well as other endogenous metabolites at m/z 78.9583, 130.9288, 248.8386, 295.8092, and 313.8187 of two-week-old and six-week-old mice brain slices were 10


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shown in Figure 2 (B) and (C), respectively.

There are two important experimental

findings: (1) Densities of different ions change with different regions.

For example,

PO3- at m/z 78.9583 shows higher densities in cerebellum while the unknown ion at m/z 130.9288 shows higher densities in olfactory bulb for both two-week-old and six-week-old mice.

Because PO3- ion can be generated from ATP, ADP,

phospholipids or phosphorylated proteins upon laser irradiation, the location of PO3provides useful information for understanding the biological processes in neurons.61 In the CA1 region of hippocampus, the combined activation of SRC family tyrosine kinases,

protein

kinase

A,

protein

kinase

C

and,

in

particular,

Ca21/calmodulin-dependent protein kinase II causes phosphorylation of glutamate receptor-gated ion channels and the enhancement of subsequent postsynaptic current.62 (2) Molecular compositions of brains changes with developmental stages. There are distinguished abundance differences of two-week-old and six-week-old mice in ions at m/z 281.2485, 309.2793 and 313.8187 which represent monounsaturated fatty acid C18:1, C20:1 and an unknown species, respectively.

So

far, biological roles of C20:1 remain largely unknown but C18:1 has been extensively studied.

Comparison of Figure 2 (B) with (C) indicates the presence of much more

branched substructures characterized with C18:1 in the brains of six-week-old mice. It has been reported that C18:1 promotes neuronal differentiation, clustering and expression of axonal growth associated proteins.63 The better developed brains of six-week-old mice shown in Figure 2 (C) is in accordance with that has been reported. Similar abundance patterns of the unknown ion at m/z 313.8187 has also been observed.

Although the identity of this ion at m/z 313.8187 is currently not available,

we believe further development of EAT-LAET in combination with other techniques may reveal even more interesting discoveries.

Attention should be paid to C20:1

that was not even detected in two-week-old mice but abundant in six-week-old mice. It is shown that brains of six-week-old mice are better developed and have relatively consistent molecular compositions within individuals.

Therefore, those mice have

been chosen for the demonstration of the electron acceptive mass tag in following experiments of mass spectrometric imaging. 11



Page 12 of 32

ex vivo ligand binding competition with endogenous Glu on mice brain tissue slices.

Because glutamate receptors are transmembrane proteins, it is very difficult

to extract and crystalize those intact proteins from cultured cells or tissues for ligand binding assays.

Autoradiography has been the well-established technique for ex vivo

ligand binding assays.

In this approach, tissue sections are incubated with

L-[3H]-glutamate with the presence of a compound to determine competed binding affinities.64, 65 Mass spectrometric imaging (MSI) provides a new experimental avenue for such studies.

It does not need radioactive reagents because those exogenous and

endogenous molecules can be directly ionized and detected by the mass analyzer. Figure 3 (A) shows the spatial distribution of endogenous Glu at m/z 146 (indicated with red squares).

Ions at m/z 130, 281 and 309 are listed as location references

because their spatial distributions clearly display brain architectures. regional variations in endogenous Glu binding sites were observed. level of glutamate was found in the olfactory bulb region.

Extensive The highest

Within the olfactory bulb,

there is a lamination with higher binding levels in the external plexiform layer.

In

addition to high levels in primary olfactory cortex, high levels of binding are also associated with the olfactory tubercles.

However, cerebral cortex exhibits both

regional and laminar variations in Glu site density. greater density than deeper layers.

In neocortex, outer layers have a

Hippocampal subfields (CA1-CA4) show

distinctive distributions of Glu binding sites.

The highest level is found in the CA1

region indicated with blue circles in Figure 3 (A) and the low levels are found over the other layers.

Midbrain and brainstem regions exhibits low levels of binding with

the exception of higher levels in the central gray.

The lowest level of glutamate

binding sites is found in the cerebellar peduncle regions (red arrows) that connect the cerebellum to the brain stem as well as the globus pallidus (blue arrows) that is involved in the regulation of voluntary movement.

Competed binding of fluorenyl tagged Glu, NMDA and their tert-butyl esters were shown in Figure 3 (B) and (C).

The irradiation with a third harmonic (355 nm) Nd3+: 12


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YAG laser results in the formation of a negative fluorenyl ion at m/z 165.0702 for all these compounds.

It was found in Figure 3 (B) that FMOC-Glu can displace almost

all the binding of endogenous Glu, except for cerebellar peduncle and olfactory tubercle regions.

High density of FMOC-Glu is found in cerebellar peduncle regions

that has the lowest density of endogenous Glu.

However, low density of FMOC-Glu

is found in olfactory tubercle region that has high density of endogenous Glu. Figure 3 (C) shows that the esterification of FMOC-Glu decreases the replacement of endogenous glutamate, especially in the cerebral cortex and vermis regions. Similarly, FMOC-NMDA also replaces the major binding of endogenous glutamate except for cerebral cortex and vermis regions but its esterification decreases the binding affinities in the olfactory region.

These experimental results demonstrate

that glutamate receptors are highly heterogeneous assemblies with different binding affinities towards different ligands.

Mass spectrometric imaging of mice brain tissue slices treated with ligands in vivo.

Mass spectrometric images of FMOC-Glu, FMOC-Glu(OtBu)-OH,

FMOC-NMDA and FMOC-NMDA(OtBu)-OH that were injected to the tail vein of mice are shown in Figure 4 and 5 respectively.

By assessing the ion at m/z 165.0702

generated from either FMOC-Glu, FMOC-Glu(OtBu)-OH, FMOC-NMDA or FMOC-NMDA(OtBu)-OH, it is demonstrated that those compounds can pass the blood-brain barrier (BBB) but reside in different regions of the brain.

While those

compounds are abundantly distributed in the majority of brain slices, they are not present in the regions that are enriched with C22:4 (m/z 331.2639), C20:1(m/z 309.2793) and C18:1(m/z 281.2481).

Comparing the mRNA expression,17 we noted

that FMOC-Glu, FMOC-NMDA and FMOC-Glu(OtBu)-OH appears in regions that have high mRNAs expression of GluN2A and GluN2B in adult brains.

Looking

back to the computational modeling, these compounds theoretically maintain their binding with GluN2A, GluN2B as well as mGluR1 and mGluR7 subunits.

The

density differences of FMOC-Glu, FMOC-Glu(OtBu)-OH, FMOC-NMDA and FMOC-NMDA(OtBu)-OH in different regions can be quantified by the ion at m/z 13



165.0702.

Page 14 of 32

For example, in slices DS 174, 177, 182 and 178, FMOC-Glu shows the

highest density than other three compounds.

FMOC-Glu(OtBu)-OH and

FMOC-NMDA(OtBu)-OH have similar low densities.

The density of FMOC-Glu is

two times higher than that of FMOC-NMDA and 3.6 times higher than that of FMOC-Glu(OtBu)-OH or FMOC-NMDA(OtBu)-OH.

It should be indicated the

local densities of compounds are also determined by in vivo ADME processes (Adsorption, Distribution, Metabolism and Excretion) in addition to bind affinities and the ability to pass through BBB.

The complexity of ADME processes directly

affects the final densities of those compounds in a specific region.

We have also

observed that different compounds cause different effects on the level of released endogenous Glu.

Compared with the blank level of endogenous Glu (shown in

Figure 3), the treatment of FMOC-Glu and FMOC-NMDA(OtBu)-OH did not significantly change the level of endogenous Glu in slices DS 170 and DS 178 and the density ratios of Glu over blank level are 1.3 and 0.9 respectively.

However, the

treatment of FMOC-Glu(OtBu)-OH and FMOC-NMDA stimulates the release of endogenous Glu in slice DS182 and DS 175 where the density ratio of Glu vs blank level is 6.6 and 1.8 respectively.

Conclusion iGluRs and mGluRs confers functional diversities and versatile binding affinities with different compounds that are pharmacologically interesting. Correlating individual synaptic signaling induced by these compounds requires a characterization of the overall spatial distribution of GluRs-targeted compounds in order to understand the concerted action of synaptic machines.

This work has developed an electron

acceptive mass tag (EAT) to covalently bond with Glu and NMDA as well as their tert-butyl esterified derives.

It functions not only as the electron acceptor to generate

mass spectrometric signals but also as the core component to block the closure of biolobed clamshell segments of GluRs.

The EAT-LAET approach provides a unique

way to monitor the dynamic distribution of tagged glutamate derivatives as well as the overall changes in other co-existing endogenous metabolites. 14


As a new mass

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spectrometric technique, it provides a strategy for quantitative analysis of different compounds with different ionization and evaporation efficiency.

As a new

pharmaceutical tool, it should be useful in drug design and disease therapy.

Methods ex vivo binding competition of tagged Glu and NMDA with endogenous Glu. Reagents and apparatus were summarized in Supplementary Notes1.

Preparation of

mice brain tissue slices and uniform thin films of (Bi2O3)0.07(CoO)0.03(ZnO)0.9 nanoparticles are described in Supplementary Notes 2.57-60 µm

thickness

were

directly

blotted

onto

Frozen tissue slices of 20 thin

films

made

of

(Bi2O3)0.07(CoO)0.03(ZnO)0.9 nanoparticles and stored in -20°C freezer for following experiments.

Tagged compounds were dissolved in a solution containing 50 mM

ammonium bicarbonate (pH 7.5) and 20% acetonitrile to reach a final concentration of 100 µM.

Slices were taken out of the freezer and thaw-mounted onto the metal

sample plate at room temperature (22°C ± 3°C ).

Ice-cold solutions of tagged

compounds were pipetted on the surfaces of tissue slices.

These slices were

incubated for 30 seconds with those solutions followed by three cycles of washing with 50 mM ammonium bicarbonate solution containing 20% acetonitrile and one cycle of washing with ethyl acetate.

Air-dried slices were subjected to mass

spectrometric imaging for the visualization of tagged compounds as well as endogenous Glu.

Blank experiments were performed in parallel with slices that

were sectioned at similar positions from different mice.

Mass spectrometric analysis and imaging.

A MALDI Synapt G2 HDMS system

(Waters, USA) mass spectrometer was used for data acquisition and imaging analysis. It is equipped with a third harmonic Nd3+: YAG high repetition laser head (355 nm) and adjustable laser beam size.

A mixture of free fatty acids including C4:0, C6:0,

C8:0, C10:0, C12:0, C14:0, C16:0, C18:0, C20:0 and C22:0 was used for mass calibration.

Imaging experiments were performed in the sensitivity mode in which

mass accuracy is 0.3 ppm.

The mixture of free fatty acids has also been put in the 15



well of lock masses for internal calibration.

Page 16 of 32

Because of the evaporation of fatty

acids, samples have been analyzed in parallel to avoid contaminations.

One is used

for mass calibration with lock masses and another one is used for data acquisition. Taken together the throughput and imaging quality, the step size has been set as 80 μm x 80 μm or 30 μm x 30 μm which takes about 10 hours to complete the imaging of a mouse brain tissue section or neuron cells.

The acquisition time for each pixel is 1

second and the laser beam size was fixed at ~15 µm.

Other instrumental parameters

including laser influx, pulse width, fire rate and pulse energy have been set as 350 units, 3 ns, 200 Hz and 100 μJ/200 Hz, respectively. negative ion mode.

All data were acquired in

The voltages on the sample plate and the aperture are set as 87

volts and 107 volts, respectively. analysis of tissue sections.

HDI software (Waters, US) was used for imaging

In order to evaluate the quantitative capability and

detection limit, 0.1 µL of a series of solutions containing 50 mM NH4HCO3 and different amounts of FMOC-Glu (OtBu)-OH and FMOC-NMDA (OtBu)-OH ranging from 0.001 mM to 200 mM was spotted on surfaces of compressed thin films of bismuth

cobalt

zinc

oxide

(Bi2O3)0.07(CoO)0.03(ZnO)0.9

nanoparticles.

Signal-to-noise ratio was set as 10 for determination of detection limit.

Because of

the spreading of the solutions, mass spectrometric intensities of each spot were integrated.

Computational methodology.

Receptor-ligand docking was performed with

Accelrys Discovery Studio 2.5 (San Diego, CA, USA).

Both ionotropic glutamate

receptors and metabotropic glutamate receptors have been computationally investigated.

Binding of non-tagged Glu and NMDA with iGluN2A was based on

the crystal structure downloaded from Protein Data Bank (code: 2A5S).

Receptor

models for tagged molecules were based on crystal structures of iGluN2A, iGluN2B, mGluR1

and

mGluR7

ligand-binding

cores

in

complex

with

PPDA

(1-(phenanthrene-2-carbonyl) piperazine-2, 3-dicarboxylic acid), Ro25-6981, and 2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-D-alaninethat (code: 4NF6, 5IOV, 3MQ4 and 3KS9).

The 3D structures of ligands were generated using 16


Page 17 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Discovery Studio.

As physiological conditions were assumed, basic aliphatic

nitrogen atoms and carboxyl groups of amino acids were protonated or de-protonated respectively.

The geometry of ligands and proteins was optimized using the

CHARMM force field and the conjugate gradient method.

For flexible docking

analysis, the initial position of the ligand in the binding sites of the receptor proteins were defined according to that reported in Protein Data Bank (code: 4BF6). Obtained docking structures were examined and selected according to the following criteria: (1) Lowest energy for the complex; (2) Localization of the hydrophobic fluorenyl group in the rear core; (3) Stabilization of the fluorenyl group in the cavity through π interactions with nearby Val, Lys and Leu amino acid residues. were visualized with Discovery Studio Visualize 2.5.

Structures

Density functional theory

(DFT) was used to study the electronic structures of molecules.

Ground state

electronic energies were calculated by using Gaussian software (Wallingford, CT, USA).

Molecular structures were first optimized and then calculated with

B3LYP/6-31G+ (d) basis set.

Charge distribution of molecules was evaluated with

natural bond orbital (NBO) calculation.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Detailed

experimental

procedures,

computational

evaluation

and

semi-quantification of binding affinities with fluorescence; binding selectivity towards different neuronal cells (PDF)

Corresponding Author Email: [email protected]

Author Contributions Ruowei Jiang performed all experiments on mass spectrometry and computations with Discovery Studio and Gaussian software.

Juan Zhang, Shanshan Jia, Weidan Li and 17



Wenting Lu performed experiments on the determination of binding affinities.

Page 18 of 32

Si

Zou, Baojie Shen and Xuekun Zou performed experiments on the culture and mass spectrometric imaging of neuronal cells. parts of the computations.

Xiebin Leng, Yinghua Qi have performed

Hongying Zhong developed the concept, designed all

experiments, analyzed all data and wrote the manuscript.

Notes of Conflict of Interest The regents of Central China Normal University have filed a patent application related to the technology described in this work to the State Intellectual Property Office of People’s Republic of China.

Hongying Zhong, Ruowei Jiang and Juan

Zhang have been listed as inventors.

Acknowledgment Authors greatly appreciate the help from Miss Jiali Li and Xiaoyuan Zhang who are an undergraduate student in School of Fine Arts of Central China Normal University. Jiali Li drew all anatomy cartons for brain tissue slices and Xiaoyuan Zhang created art works.

We greatly appreciate the support from National Natural Science

Foundation of China (NSFC, 21575046 and 81761128005), Research Funds of Central China Normal University from the Ministry of Education, The Program of Introducing Talents of Discipline to Universities of China (111 program, B17019), International Joint Research Center for Intelligent Biosensing Technology and Health, Hubei International Scientific and Technological Cooperation Base of Pesticide and Green Synthesis.

18


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Monaghan,

D.

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25



Page 26 of 32

Figure Legend

Figure 1. Design of the electron acceptive mass tag. capture ionization and dissociation. different compounds.

(A) Diagram of photoelectron

(B) Generation of the ion at m/z 165.0702 from

V0, V1, V2 and V3 are voltages applied to the sample plate,

extraction plate, hexapole and aperture, respectively.

LBD represents the ligand

binding domain.

Figure 2. Mass spectrometric imaging of endogenous metabolites in mice brains without the treatment of any exogenous chemicals.

(A) Preparation of mice brain

slices and a representative mass spectrum of a slice.

(B)

mouse brain slices.

Images of two-week-old

(C) Images of six-week-old mouse brain slices.

DSi (i=1 to n)

represents slice codes that were counted from dorsal aspect to ventral aspect in every 20 μm step.

Fatty acids indicated with stars are used for site references.

Figure 3. Ex vivo ligand binding competition of tagged compounds with endogenous Glu.

(A) Brain slices without treatment.

FMOC-Glu

and

FMOC-NMDA.

(C)

(B) Brain slices incubated with Brain

FMOC-Glu(OtBu)-OH and FMOC-NMDA(OtBu)-OH.

slices

incubated

with

Ions at m/z 130.9288,

281.2481 and 309.2793 were used for site references.

Figure 4. Mass spectrometric imaging of mice brains that have been in vivo tail vein injected with (A) FMOC-Glu and (B) FMOC-Glu(OtBu)-OH.

DSi (i=1 to n)

represents the slice codes that were counted from dorsal aspect to ventral aspect in every 20 μm step.

Figure 5. Mass spectrometric imaging of mice brains that have been in vivo tail vein injected with (A) FMOC-NMDA and (B) FMOC-NMDA(OtBu)-OH.

DSi (i=1 to n)

represents the slice codes that were counted from dorsal aspect to ventral aspect in every 20 μm step. 26


Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(A)

Mass Analyzer V3 V2 V1 Radical anion

GluN1 GluN2A

Glu/NMDA electron acceptor linker

Fragments

Zinc

S1

photoexcited electrons

Glycine

LBD

e-

S2 Extracellular

Laser irradiation (355 nm)

Intracellular semiconductor nanoparticles V0

Sample Plate

(B) FMOC-Glu

Negative fluorenyl ion at m/z 165.0702 O

eO

O O

HO

O O

+

NH

O

NH

HO OH

O

N

N

O

O

O

HO

HO

+O

O OH

OH O

OH

m/z 165.0702 O

O O

O O

OH

e-

FMOC-NMDA

NH

O

HO

O

O

Figure 1

27


N

HO

O OH

O


%

(A)

180.9038

100 80

Page 28 of 32

Vermis

196.8883

78.9583

Cerebral peduncle

☆

60

Pituitary stalk

255.2324

☆

168.9318

40

Olfactory tubercle Cerebrum

281.2485

☆

Neocortex

283.2639

20

Cerebellum Pineal gland

Olfactory bulb

Olfactory bulb

0 100

150

200

250

300

350

400

m/z

DSn

DS1

Ventral side

Dorsal side

(B)

Two-week-old

DS153

DS156:

PO3-

PO3-

m/z 130.9288

m/z 248.8386

m/z 295.8092

C18:1

C20:1

m/z 313.8187

m/z 130.9288

m/z 248.8386

m/z 295.8092

C18:1

C20:1

m/z 313.8187

Six-week-old

DS177

DS180:

PO3-

PO3-

(C)

m/z 130.9288

m/z 248.8386

m/z 295.8092

C18:1

C20:1

m/z 313.8187

m/z 130.9288

m/z 248.8386

m/z 295.8092

C18:1

C20:1

m/z 313.8187

Figure 2

28


Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Endogenous Glu without treatment

DS168: m/z 130.9288

Glu

(A)

C18:1

C20:1

DS182: m/z 130.9288

Competition of FMOC-Glu

DS177: Fluorenyl

C18:1

C20:1

(B)

Competition of FMOC-NMDA

Glu

C18:1

C20:1

DS180: Fluorenyl

Competition of FMOC-Glu(OtBu)-OH

DS168: Fluorenyl

Glu

Glu

Glu

C18:1

C20:1

Competition of FMOC-NMDA(OtBu)-OH

C18:1

C20:1

DS169: Fluorenyl

Figure 3

29


Glu

(C)

C18:1

C20:1


Page 30 of 32

(A)

FMOC-Glu treated

DS170: C18:1

DS170: m/z 130.9288

C20:1

Glu

C22:4

DS174: C18:1

DS174: m/z 130.9288

Fluorenyl

C20:1

Glu

C22:4

Fluorenyl

FMOC-Glu(OtBu)-OH treated

DS177: C18:1

DS177: m/z 130.9288

(B)

C20:1

C22:4

DS182: C18:1

Glu

Fluorenyl

DS182: m/z 130.9288

Figure 4

30


C20:1

Glu

C22:4

Fluorenyl

Page 31 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(A)

FMOC-NMDA treated

DS176: C18:1

DS176: m/z 130.9288

C20:1

C22:4

Glu

Fluorenyl

DS182: C18:1

DS182: m/z 130.9288

C20:1

Glu

C22:4

Fluorenyl

FMOC-NMDA(OtBu)-OH treated

DS178: C18:1

DS178: m/z 130.9288

C20:1

Glu

(B)

C22:4

DS183: C18:1

Fluorenyl

DS183: m/z 130.9288

Figure 5

31


C20:1

Glu

C22:4

Fluorenyl


135x124mm (150 x 150 DPI)


Page 32 of 32

An Electron Acceptive Mass Tag for Mass Spectrometric Imaging

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