Mass SPectrometr Y IMS Ana LYs Is o F Neurotransm Itters DOI: 10.5702/massspectrometry.S0049
Vol. 3 (2014), S0049
Review
Imaging Mass Spectrometric Analysis of Neurotransmitters: A Review Gustavo A. Romero-Perez, Shiro Takei, and Ikuko Yao* Hamamatsu University School of Medicine, 1–20–1 Handayama, Higashi-ku, Hamamatsu 431–3192, Japan
Imaging mass spectrometry (IMS) is a toolbox of versatile techniques that enable us to investigate analytes in samples at molecular level. In recent years, IMS, and especially matrix-assisted laser desorption/ionisation (MALDI), has been used to visualise a wide range of metabolites in biological samples. Simultaneous visualisation of the spatial distribution of metabolites in a single sample with little tissue disruption can be considered as one important advantage of MALDI over other techniques. However, several technical hurdles including low concentrations and rapid degradation rates of small molecule metabolites, matrix interference of signals and poor ionisation, need to be addressed before MALDI can be considered as a reliable tool for the analysis of metabolites such as neurotransmitters in brain tissues from different sources including humans. In the present review we will briefly describe current MALDI IMS techniques used to study neurotransmitters and discuss their current status, challenges, as well as future prospects. Please cite this article as: G. A. Romero-Perez, et al., Imaging Mass Spectrometric Analysis of Neurotransmitters: A Review, Mass Spectrom (Tokyo) 2014; 3(4): S0049; DOI: 10.5702/massspectrometry.S0049 Keywords: imaging mass spectrometry, IMS, neurotransmitter, MALDI (Received October 27, 2014; Accepted January 22, 2015)
INTRODUCTION Imaging mass spectrometry (IMS) techniques provide an unparalleled approach to study the molecular makeup of tissues whilst preserving critical information on the spatial distribution of analytes such as lipids,1,2) peptides and proteins,3,4) amino acids,5) and other small molecules. During IMS analysis, pre-selected positions create an array of points separated from each other at a determined distance and a mass spectrum is obtained at each spot.6,7) With the development of automated instruments8,9) and powerful software,10,11) capable of rapidly acquiring and processing orderly yet massive imaging spectral data, it is now possible to simultaneously visualise innumerable individual biomolecules in tissue sections.7,10) The identification and localisation of compounds such as neuropeptides,12,13) neurotransmitters,14) and small molecules2) are especially important for the study of the brain, as it is a complex organ with a high spatial and somatotopic organisation. An outstanding feature of IMS is the ability to analyse hundreds of metabolites in a single run without the need of prior labelling or knowledge of the metabolites whilst providing both spatial and mass information.7) Currently, there are three major ionisation methods used for IMS analysis: secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption/ionisation MS (MALDI MS) and desorption electrospray ionisation (DESI), and all their variants. A summary of recently reported neurotransmitter analyses by these techniques is shown in Table 1. In the following pages we will concisely review the principle of IMS analysis with
emphasis on MALDI, and the current status of neurotransmitters analysis by this technique. Also, current limitations and challenges as well as future outlook of IMS techniques will be briefly discussed.
MATRIX-ASSISTED LASER DESORPTION/ IONISATION (MALDI) Matrix-assisted laser desorption/ionisation (MALDI) is currently regarded as the most popular IMS technique and one of the two “soft” ionisation methods used to analyse samples, the other being electrospray ionisation (ESI)15) (Table 1). As Römpp and Spengler explained in a recent review, for MALDI MS analysis, tissue samples need to be coated with a chemical compound acting as matrix. This matrix permits desorption and ionisation of samples metabolites by a laser beam irradiated onto a defined region of the sample.16) Ions are then drawn into a mass spectrometer, where flight time is recorded and converted to mass-to-charge (m/z) values, typically covering a range over 50,000 Da.17) Also, the surface of the sample is scanned in a raster by a nanosecond laser beam. This process is repeated at pre-determined positions and a mass spectrum is produced. Thousands of spectra are then sequentially acquired in this manner, which generates a molecular profile for a pre-selected region of tissue.7) One important advantage of MALDI over other analytical methods is that little disruption of the tissue occurs, which enables the retention of the spatial distribution of metabolites such as proteins and peptides in the sample.18) Nonetheless, regarding the analysis of neurochemicals by MALDI, one critical concern is
* Correspondence to: Ikuko Yao, Department of Optical Imaging, Medical Photonics Center, Hamamatsu University School of Medicine, 1–20–1 Handayama, Higashiku, Hamamatsu 431–3192, Japan, e-mail: [email protected]
© 2014 The Mass Spectrometry Society of Japan
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its quantitative ability. Indeed, more often than not, peaks from matrices share a similar low-mass range as those from many endogenous metabolites including neurotransmitters. As a result, ion intensity obtained by MALDI analysis is difficult to interpret, and mass-assigned peaks to predetermined compounds, as well as validation by MS/MS analysis are necessary.14,19) Another concern that can be mentioned is its limited sensitivity for detecting trace amounts of biomolecules in tissues samples. This is believed to be caused by strong ion suppression effects due to the lack of sample purification procedures.20) These technical hurdles have somewhat limited the capacity of MALDI as a reliable tool in neurochemical research.
IMAGING MASS SPECTROMETRY OF NEUROTRANSMITTERS Neurotransmitters are endogenous molecules that carry signals from a neuron to a specific cell along a synapse.21,22) They are also believed to be closely involved in the regulation of several processes such as the immune system and metabolism.23) Depending on their structure and chemical properties, these biomolecules can be found in different compound groups including peptides, amino acids and amines24) (Tables 1 and 2). The study of neurotransmitters has enabled us to understand the relationship between the biochemical processes in the central nervous system (CNS)
Table 1. Summary of current literature on neurotransmitter analysis by major imaging mass spectrometry techniques. Optimal analytes
Biological source
Tissue
Ionisation type
IMS technique
Reference
Soft ionisation Amino acids Glutamate, glycine
Mouse
Liver
MALDI TOF/TOF
5
GABA, glutamate, tyrosine
Mouse, rat, rhesus monkey
Brain
MALDI FT-ICR
7a, 54
Mouse
Spinal cord
MALDI TOF/TOF
18
Mouse, rat
Brain
MALDI FT-ICR
54–56
Dopamine
Swine
Adrenal gland
MALDI FT-ICR
7
Dopamine, 3-MT, serotonin
Ratb, rhesus monkey
Brain
MALDI FT-ICR
54
Adenosine, AMP, ADP, ATP
Rat, mouse
Brain
MALDI TOF/TOF
6
Epinephrine, norepinephrine
Swine
Adrenal gland
MALDI FT-ICR
7
Norepinephrine, epinephrine
Swine
Adrenal gland
DESI
9
TOF-SIMS
63d
Small molecule metabolites Acetylcholine
Hard ionisation Opioid, amyloid peptidesc
—
—
Glutamate and tyrosine analyses were not conducted by these workers. bRat brain tissues were not imaged for serotonin. cSynthetic peptides. Peptides were spotted onto the surface of a silicon wafer substrate. GABA: γ-aminobutyric acid, 3-MT: 3-methoxytyramine, AMP: Adenosine monophosphate, ADP: Adenosine diphosphate, ATP: Adenosine triphosphate, IMS: Imaging mass spectrometry, MALDI: Matrix assisted laser desorption/ionisation, TOF: Time of flight, FT-ICR: Fourier transform ion cyclotron resonance, DESI: Desorption electrospray ionisation, SIMS: Secondary ion mass spectrometry. a
d
Table 2. Optimal analytes
Summary of visualised neuropeptide by IMS. Biological source
Tissue sample
Reference
Dynorphin B alpha-Neoendorphin
Rat
Brain
16
FMRFamide-related peptides SIFamide Crustacean hyperglycaemic hormones Orcokinin-related peptides Tachykinin-related peptides AST-A Pigment-dispersing hormone
Black tiger prawn
Eyestalk, brain, thoracic ganglia
41
RFamides Orcokinins AST-B Orcomyotropin-related peptide SIFamide CabTRP 1a Pyrokinin
Jonah crab
Brain
42
AmTRP-5 AST-1
Africanized honeybee
Brain
43
© 2014 The Mass Spectrometry Society of Japan
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and the homeostatic state of behaviour, cognition and mood.25) Aberrant neurotransmission is associated with conditions such as depression,26,27) drug abuse28) and degenerative diseases.13,22) Insofar the analysis of neurotransmitters has had a slow progress, mainly due to technical limitations. For example, Perry et al. explain that, even though microelectrodes and biosensors capillary electrophoresis (CE) have been commonly used to quantitate neurotransmitters in the extracellular space of tissues, analyses by this equipment are hindered by the size, complexity and fragility of samples, low selectivity measurements and spatial resolution, and electroactivity of other neurotransmitters, amongst others.25) In contrast, IMS is regarded as a promising alternative, as it can provide higher sensitivity, selectivity and signal-to-noise (S/N) during the analysis of neurotransmitters.14) In the following sections we will briefly discuss the IMS techniques used to analyse major neurotransmitter classes.
Imaging mass spectrometry techniques for neuropeptides
Neuropeptides are relatively large signalling biomolecules constituted by three to 70 amino acids.29) Neuropeptides can be either excitatory or inhibitory neurotransmitters and thus, play seemingly antagonistic roles in various conditions, from euphoria30) and pain reduction,31) to anxiety32) and panic attacks.33) In the last 40 years, mass spectrometry has been a reliable tool to identify and quantify neuropep-
Fig. 1.
Vol. 3 (2014), S0049
tides in samples from various sources.34–36) Nonetheless, more recently MALDI IMS has become the method of choice to characterise neuropeptides in brain tissues of crustaceans,37,38) insects,39) rodents12) and human.40) Yet, there are several recurring problems surrounding MALDI analyses. One such problem is the need for proper sample preparation to minimise rapid molecular degradation.29) In addition, most of the time matrices are required for MALDI analyses, but its use renders sample preparation more elaborate, and prevents the analyses of samples in their native form. Matrix selection and deposition on samples are critical to obtain high sensitivity, spatial resolution and specific ionisation of sample analytes.4,7) The size of the matrix crystals is critical for a good spatial resolution, but common matrices form large crystals and distribute non-uniformly across the sample surface.41) Moreover, the number of available matrices exhibiting light absorbance and proton donation—necessary features to achieve both high sensitivity and sample ionisation—is still very small.42) In addition, the presence in tissues samples of other biomolecules such as lipids can hinder visualisation of the neuropeptide distribution in intact, untreated brain tissues, due to the interference of signals. To extract lipids and improve the detection of neuropeptides, brain tissue sections can be washed with organic solvents,43) but this procedure can relocate proteins and peptides. The summary of visualised neuropeptide by IMS is shown in Table 2. Nonetheless, as Chansela et al. report, using brain tissues previously prepared with paraffin-
Imaging by MALDI of paraffin-embedded sections of eyestalk, brain and thoracic ganglia tissues of shrimp Penaeus monodon. Composite Fig. 1A depicts a paraffin-embedded section (PETS) of P. monodon eyestalk tissue (upper left image) after hematoxylin–eosin (H&E) staining where the magnified structures of the retina (R), the lamina ganglionalis (LG), the medulla externa (ME), the medulla interna (MI) and the medulla terminalis (MT) can be seen. Under similar treatment conditions as above, the middle left image in Fig. 1A is a PETS of P. monodon brain tissue that shows the magnified structures of the olfactory lobe (OL) and the accessory neuropil (AL), and the lower left image is a PETS of P. monodon thoracic ganglia tissue with the magnified structure of the neuropil. The ion intensity maps of neuropeptides Phenylalanine-Methionine-Arginine-Phenylalanine (FMRF) and AYRKPPFNGSIF (SIF) amides can be seen Figs. 1B and 1C, which show that the strongest signals were detected in P. monodon thoracic ganglia and brain tissues, but especially in clusters 11 and 14 of the brain (white arrowheads). In contrast, at m/z 1338.6, crustacean hyperglycaemic hormone molecules (CHH) were found only in low levels in ME and MI of ES, the thoracic ganglia and clusters 11 and 14 of the brain (3D). Images and text adapted from Chansela et al., 2012, with permission.
© 2014 The Mass Spectrometry Society of Japan
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Fig. 2.
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Spectral and imaging data by MALDI MS of γ-aminobutyric acid, dopamine, and 3-methoxytyramine distributions in tissues sections of rat brain. (A) The composite spectrum generated by MALDI shows the individual peaks of γ-aminobutyric acid (GABA), catecholamine dopamine (DA) and 3-methoxytyramine (3-MT). After in situ derivatisation with 2,4-diphenyl-pyranylium tetrafluroborate (DPP-TFB), Shariatgorji et al. successfully visualised in sagittal sections of rat brain tissue (B), the relative abundance and distribution of GABA (C), DA (D), and 3-MT (E). GABA was found to be most abundant in the hypothalamus (HY) and the basal forebrain (BF), whereas DA (D) and 3-MT in the stantia nigra (SN), the caudate-putamen (CP), the ventral striatum (STRv) and the anterior olfactory nucleus (AON). Images and text adapted from Shariatgorji et al., 2014, with permission.
embedded tissue sections (PETS) greatly help to image neuropeptides by MALDI IMS, because histological structures are preserved and acetone or ethanol can be used to remove lipids by dehydration.37) Chansela et al. successfully visualised by MALDI IMS the distribution of CNS neuropeptides in PETS of shrimp Panaeusmonodon, with no reported analytical problems37) (Fig. 1). In a similar manner, after embedding brain tissues of crab Cancer borealis in paraffin blocks, Chen et al. used a 3D mapping technique to localise the distribution of several neuropeptides.38)
Imaging mass spectrometry techniques for amino acid neurotransmitters
Some amino acids can act as neurotransmitters by mediating the signalling between neurons, and between neuron and glia cells. Glutamate, γ-aminobutyric acid (GABA), and serine, amongst others, are considered amino acids capable of transmitting these signals.29) In the classic view, glutamate is the major mediator of excitatory signalling in the CNS,44) whereas GABA is an inhibitory neurotransmitter.45) Nonetheless, Chavas and Marty46) recently showed that a dual role (inhibition and excitation) of GABA was found in the cerebellar interneuron network. In contrast, serine is involved in long-term potentiation, fear conditioning and nociception, and abnormal levels of this amino acid have been linked to chronic neurodegenerative disease and Alzheimer’s disease.47) Although glutamate and GABA have been extensively analysed by chromatography techniques,48,49) IMS analysis of amino acid neurotransmitters is still an incipient area of research. Weak ionisation and interference by ion signals of matrices during IMS detection © 2014 The Mass Spectrometry Society of Japan
can be listed as the technical challenges that have prevented visualisation and quantification of amino acid neurotransmitters by IMS.5) With modifications to pre-column derivatisation and HPLC/ESI-tandem MS techniques previously reported by Nakatsukasa et al.,50) Toue at al. developed a derivatisation procedure for sections of murine tumourbearing liver tissues and analysed them by MALDI IMS to obtain semi-quantitative spatial information on several amino acids including glutamate, GABA and serine.5) Using an ingenious approach, Sugiura et al. combined IMS with focused microwave irradiation—which effectively stopped and preserved in vivo brain metabolism in brain tissues—to quantify and map glutamate in glutaminergic neuron-rich regions.51) Furthermore, very recently Shariatgorji et al. reported a promising technique for the direct and absolute quantitative imaging of neurotransmitters including GABA and glutamate, which addresses not only the interference by matrix signals but also the poor ionisation efficiency by MALDI52) (Fig. 2).
Imaging mass spectrometry techniques for small molecule neurotransmitters
Acetylcholine (ACh) was the first compound to be defined as neurotransmitter and its primary role is to act as an excitatory neurotransmitter in muscular synapses, except in the heart, where it inhibits signal transmission.25,29) Abnormal ACh signalling has been associated with Huntington’s, Alzheimer’s and Parkinson’s diseases, and schizophrenia.29) There are only few, but nevertheless critical technical impediments that complicate ACh analysis by IMS. For example, in a recent experiment, we underlined the fact Page 4 of 9
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Fig. 3.
Vol. 3 (2014), S0049
Tandem IMS analysis of acetylcholine in tissue sections of murine spinal cord. Our laboratory successfully imaged the spatial distribution of acetylcholine (ACh) in murine spinal cord tissues by conducting tandem IMS analysis. Figure 3A shows the average spectra of the total product ion data generated by IMS. ACh-derived product ion was detected at m/z 87 (inset), which was obtained as a neutral loss of a trimethyl-amine group (NL59) from the original intact ion at m/z 146. Figure 3B shows the intensity ion map after reconstruction and Fig. 3C depicts the final ion distribution map of galactosylceramide after merging. Arrowheads (3C) show cholinergic motor neuron-containing regions in the murine spinal cord where ACh was found to be most abundant. Images and text adapted from Sugiura et al., 2012, with permission.
that, since ACh exhibits a high degradation rate, an efficient sample preparation was needed to minimise post-mortem deterioration.14) To that end, we used liquid nitrogen to flash-freeze brains tissues of deeply anaesthetised mouse, a technique known as in situ freezing, which minimised postmortem degradation whilst improving dynamic range and sensitivity. We also found that interference by the matrix signals to that of the precursor ion was another problem. We successfully avoided this by detecting ion transitions in tandem MS14) (Fig. 3). The use of common matrices such as DHB and CHCA is yet another technical hindrance, as these compounds usually cause interference for ACh detection by IMS. Shariatgorji et al. avoided matrix interference during the MALDI analysis of ACh by using the deuterated form of CHCA (D4-CHCA). Matrix D4-CHCA overcomes interference by shifting signals by a minimum of 4 m/z units.53) In a different approach, Ye et al. tested the resolution capacity of MALDI LTQ Orbitrap XL MS, and found that it was powerful enough to separate analyte signals even from those of traditional matrices. Indeed, using MALDI LTQ Orbitrap XL MS, Ye et al. accurately mapped ACh in mouse brain tissues coated with DHB as matrix.54) Serotonin (5HT), dopamine (DA), norepinephrine (NE), © 2014 The Mass Spectrometry Society of Japan
and epinephrine (EP) are amines that act as neurotransmitters, of which DA is considered the most abundant amongst them.25) Previous data show that DA pathways are associated with perception of reward and mediation of learning and feeding, and thus addictions are strongly related to the dopamine system.25) Similarly, NE and EP are thought to be neurotransmitters with excitatory roles in brain functions related to arousal, attention, learning, memory and stress response.55) In contrast, 5HT regulates brain functions during alertness and motor activity as well as primal behaviours such as eating, sleeping and reproduction.56) Nevertheless, disorders of the 5HT systems have been associated with depression, anxiety, and schizophrenia.25) Imaging MS analysis of these amine transmitters has been limited by similar challenges as those faced by IMS amino acid analysis, although a few experiments have achieved certain success. For example, NE and EP levels were detected in the adrenal gland of adult pigs by IMS without matrix.57) Likewise, visualisation and quantitation of dopamine in brain tissue by MALDI IMS was possible by derivatising dopamine with 2,4-diphenyl pyrylium using CHCA as matrix.29) Purine ring-containing nucleosides adenosine, AMP, ADP, and ATP are signalling molecules of about 300– 1000 Da that have been found to act as neurotransmitters.29) Page 5 of 9
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Fig. 4.
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Imaging by MALDI IMS of in situ distribution of adenosine, adenosine monophosphate, adenosine diphosphate and adenosine triphosphate in control mice and ischemic rats. Images show in situ changes in abundance of adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) during ATP metabolism in coronally-sectioned brain tissues of control and ischemic rodent models, after being subjected for 24 h to middle cerebral artery occlusion. The adenosine structure was identified by comparing MS or tandem MS spectra with those of a standard compound. Scale bar: 1 mm. Images and text adapted from Liu et al., 2014, with permission. ISCH: tissue images of ischemic rats, CON: tissue images of control mice.
Adenosine and ATP are excitatory neurotransmitters in the nervous system that mediate signalling to motor neurons in the spinal cord and autonomic ganglia.25,29) As with most small molecules, attempts to measure and visualise purine neurotransmitters by IMS have been hindered by rapid degradation rates, low concentrations and matrix-related signal interference. In a ground-breaking work, Suematsu and colleagues successfully mapped the distribution of UDP,51) and AMP, ADP, and ATP58) in mouse brain tissues. To achieve this, they first minimised post-mortem degradation of tissue samples by either in situ freezing58) or irradiating them with focused microwave—which has been discussed above.51) Next these workers used stand-alone MALDI IMS,51) or MALDI IMS combined with CE-ESI-MS,58) to quantify and visualise metabolic fluxes of UDP-glucose complex, and to construct maps of adenine nucleotides for identification of AMP, ADP, and ATP in absolute terms (e.g., µM/g of brain tissue), respectively. In a more recent work, Liu et al. deposited 1,5-naphthalenediamine (1,5-DAN) hydrochloride onto the brain tissue sections of control and ischemic rodent models, using an automatic matrix sprayer, which permitted a homogeneous matrix coating of the whole tissue surface. Matrix 1,5-DAN showed strong ultraviolet absorption, high tolerance to salts and fewer background signals, especially in the low mass range (m/z 500 or lower).59) With this approach, Liu et al. were able to successfully map by MALDI IMS the distribution of adenosine, AMP, ADP, and ATP in the tissue sections, and estimate the changes of these metabolites during ATP metabolism58,59) (Table 1, Fig. 4).
© 2014 The Mass Spectrometry Society of Japan
SECONDARY ION MASS SPECTROMETRY, A COMPLEMENTARY TECHNIQUE TO MALDI Secondary ion mass spectrometry (SIMS) is a hard ionisation IMS technique that, unlike MALDI, targets elements, and small mass compounds (
Vol. 3 (2014), S0049
Review
Imaging Mass Spectrometric Analysis of Neurotransmitters: A Review Gustavo A. Romero-Perez, Shiro Takei, and Ikuko Yao* Hamamatsu University School of Medicine, 1–20–1 Handayama, Higashi-ku, Hamamatsu 431–3192, Japan
Imaging mass spectrometry (IMS) is a toolbox of versatile techniques that enable us to investigate analytes in samples at molecular level. In recent years, IMS, and especially matrix-assisted laser desorption/ionisation (MALDI), has been used to visualise a wide range of metabolites in biological samples. Simultaneous visualisation of the spatial distribution of metabolites in a single sample with little tissue disruption can be considered as one important advantage of MALDI over other techniques. However, several technical hurdles including low concentrations and rapid degradation rates of small molecule metabolites, matrix interference of signals and poor ionisation, need to be addressed before MALDI can be considered as a reliable tool for the analysis of metabolites such as neurotransmitters in brain tissues from different sources including humans. In the present review we will briefly describe current MALDI IMS techniques used to study neurotransmitters and discuss their current status, challenges, as well as future prospects. Please cite this article as: G. A. Romero-Perez, et al., Imaging Mass Spectrometric Analysis of Neurotransmitters: A Review, Mass Spectrom (Tokyo) 2014; 3(4): S0049; DOI: 10.5702/massspectrometry.S0049 Keywords: imaging mass spectrometry, IMS, neurotransmitter, MALDI (Received October 27, 2014; Accepted January 22, 2015)
INTRODUCTION Imaging mass spectrometry (IMS) techniques provide an unparalleled approach to study the molecular makeup of tissues whilst preserving critical information on the spatial distribution of analytes such as lipids,1,2) peptides and proteins,3,4) amino acids,5) and other small molecules. During IMS analysis, pre-selected positions create an array of points separated from each other at a determined distance and a mass spectrum is obtained at each spot.6,7) With the development of automated instruments8,9) and powerful software,10,11) capable of rapidly acquiring and processing orderly yet massive imaging spectral data, it is now possible to simultaneously visualise innumerable individual biomolecules in tissue sections.7,10) The identification and localisation of compounds such as neuropeptides,12,13) neurotransmitters,14) and small molecules2) are especially important for the study of the brain, as it is a complex organ with a high spatial and somatotopic organisation. An outstanding feature of IMS is the ability to analyse hundreds of metabolites in a single run without the need of prior labelling or knowledge of the metabolites whilst providing both spatial and mass information.7) Currently, there are three major ionisation methods used for IMS analysis: secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption/ionisation MS (MALDI MS) and desorption electrospray ionisation (DESI), and all their variants. A summary of recently reported neurotransmitter analyses by these techniques is shown in Table 1. In the following pages we will concisely review the principle of IMS analysis with
emphasis on MALDI, and the current status of neurotransmitters analysis by this technique. Also, current limitations and challenges as well as future outlook of IMS techniques will be briefly discussed.
MATRIX-ASSISTED LASER DESORPTION/ IONISATION (MALDI) Matrix-assisted laser desorption/ionisation (MALDI) is currently regarded as the most popular IMS technique and one of the two “soft” ionisation methods used to analyse samples, the other being electrospray ionisation (ESI)15) (Table 1). As Römpp and Spengler explained in a recent review, for MALDI MS analysis, tissue samples need to be coated with a chemical compound acting as matrix. This matrix permits desorption and ionisation of samples metabolites by a laser beam irradiated onto a defined region of the sample.16) Ions are then drawn into a mass spectrometer, where flight time is recorded and converted to mass-to-charge (m/z) values, typically covering a range over 50,000 Da.17) Also, the surface of the sample is scanned in a raster by a nanosecond laser beam. This process is repeated at pre-determined positions and a mass spectrum is produced. Thousands of spectra are then sequentially acquired in this manner, which generates a molecular profile for a pre-selected region of tissue.7) One important advantage of MALDI over other analytical methods is that little disruption of the tissue occurs, which enables the retention of the spatial distribution of metabolites such as proteins and peptides in the sample.18) Nonetheless, regarding the analysis of neurochemicals by MALDI, one critical concern is
* Correspondence to: Ikuko Yao, Department of Optical Imaging, Medical Photonics Center, Hamamatsu University School of Medicine, 1–20–1 Handayama, Higashiku, Hamamatsu 431–3192, Japan, e-mail: [email protected]
© 2014 The Mass Spectrometry Society of Japan
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its quantitative ability. Indeed, more often than not, peaks from matrices share a similar low-mass range as those from many endogenous metabolites including neurotransmitters. As a result, ion intensity obtained by MALDI analysis is difficult to interpret, and mass-assigned peaks to predetermined compounds, as well as validation by MS/MS analysis are necessary.14,19) Another concern that can be mentioned is its limited sensitivity for detecting trace amounts of biomolecules in tissues samples. This is believed to be caused by strong ion suppression effects due to the lack of sample purification procedures.20) These technical hurdles have somewhat limited the capacity of MALDI as a reliable tool in neurochemical research.
IMAGING MASS SPECTROMETRY OF NEUROTRANSMITTERS Neurotransmitters are endogenous molecules that carry signals from a neuron to a specific cell along a synapse.21,22) They are also believed to be closely involved in the regulation of several processes such as the immune system and metabolism.23) Depending on their structure and chemical properties, these biomolecules can be found in different compound groups including peptides, amino acids and amines24) (Tables 1 and 2). The study of neurotransmitters has enabled us to understand the relationship between the biochemical processes in the central nervous system (CNS)
Table 1. Summary of current literature on neurotransmitter analysis by major imaging mass spectrometry techniques. Optimal analytes
Biological source
Tissue
Ionisation type
IMS technique
Reference
Soft ionisation Amino acids Glutamate, glycine
Mouse
Liver
MALDI TOF/TOF
5
GABA, glutamate, tyrosine
Mouse, rat, rhesus monkey
Brain
MALDI FT-ICR
7a, 54
Mouse
Spinal cord
MALDI TOF/TOF
18
Mouse, rat
Brain
MALDI FT-ICR
54–56
Dopamine
Swine
Adrenal gland
MALDI FT-ICR
7
Dopamine, 3-MT, serotonin
Ratb, rhesus monkey
Brain
MALDI FT-ICR
54
Adenosine, AMP, ADP, ATP
Rat, mouse
Brain
MALDI TOF/TOF
6
Epinephrine, norepinephrine
Swine
Adrenal gland
MALDI FT-ICR
7
Norepinephrine, epinephrine
Swine
Adrenal gland
DESI
9
TOF-SIMS
63d
Small molecule metabolites Acetylcholine
Hard ionisation Opioid, amyloid peptidesc
—
—
Glutamate and tyrosine analyses were not conducted by these workers. bRat brain tissues were not imaged for serotonin. cSynthetic peptides. Peptides were spotted onto the surface of a silicon wafer substrate. GABA: γ-aminobutyric acid, 3-MT: 3-methoxytyramine, AMP: Adenosine monophosphate, ADP: Adenosine diphosphate, ATP: Adenosine triphosphate, IMS: Imaging mass spectrometry, MALDI: Matrix assisted laser desorption/ionisation, TOF: Time of flight, FT-ICR: Fourier transform ion cyclotron resonance, DESI: Desorption electrospray ionisation, SIMS: Secondary ion mass spectrometry. a
d
Table 2. Optimal analytes
Summary of visualised neuropeptide by IMS. Biological source
Tissue sample
Reference
Dynorphin B alpha-Neoendorphin
Rat
Brain
16
FMRFamide-related peptides SIFamide Crustacean hyperglycaemic hormones Orcokinin-related peptides Tachykinin-related peptides AST-A Pigment-dispersing hormone
Black tiger prawn
Eyestalk, brain, thoracic ganglia
41
RFamides Orcokinins AST-B Orcomyotropin-related peptide SIFamide CabTRP 1a Pyrokinin
Jonah crab
Brain
42
AmTRP-5 AST-1
Africanized honeybee
Brain
43
© 2014 The Mass Spectrometry Society of Japan
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and the homeostatic state of behaviour, cognition and mood.25) Aberrant neurotransmission is associated with conditions such as depression,26,27) drug abuse28) and degenerative diseases.13,22) Insofar the analysis of neurotransmitters has had a slow progress, mainly due to technical limitations. For example, Perry et al. explain that, even though microelectrodes and biosensors capillary electrophoresis (CE) have been commonly used to quantitate neurotransmitters in the extracellular space of tissues, analyses by this equipment are hindered by the size, complexity and fragility of samples, low selectivity measurements and spatial resolution, and electroactivity of other neurotransmitters, amongst others.25) In contrast, IMS is regarded as a promising alternative, as it can provide higher sensitivity, selectivity and signal-to-noise (S/N) during the analysis of neurotransmitters.14) In the following sections we will briefly discuss the IMS techniques used to analyse major neurotransmitter classes.
Imaging mass spectrometry techniques for neuropeptides
Neuropeptides are relatively large signalling biomolecules constituted by three to 70 amino acids.29) Neuropeptides can be either excitatory or inhibitory neurotransmitters and thus, play seemingly antagonistic roles in various conditions, from euphoria30) and pain reduction,31) to anxiety32) and panic attacks.33) In the last 40 years, mass spectrometry has been a reliable tool to identify and quantify neuropep-
Fig. 1.
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tides in samples from various sources.34–36) Nonetheless, more recently MALDI IMS has become the method of choice to characterise neuropeptides in brain tissues of crustaceans,37,38) insects,39) rodents12) and human.40) Yet, there are several recurring problems surrounding MALDI analyses. One such problem is the need for proper sample preparation to minimise rapid molecular degradation.29) In addition, most of the time matrices are required for MALDI analyses, but its use renders sample preparation more elaborate, and prevents the analyses of samples in their native form. Matrix selection and deposition on samples are critical to obtain high sensitivity, spatial resolution and specific ionisation of sample analytes.4,7) The size of the matrix crystals is critical for a good spatial resolution, but common matrices form large crystals and distribute non-uniformly across the sample surface.41) Moreover, the number of available matrices exhibiting light absorbance and proton donation—necessary features to achieve both high sensitivity and sample ionisation—is still very small.42) In addition, the presence in tissues samples of other biomolecules such as lipids can hinder visualisation of the neuropeptide distribution in intact, untreated brain tissues, due to the interference of signals. To extract lipids and improve the detection of neuropeptides, brain tissue sections can be washed with organic solvents,43) but this procedure can relocate proteins and peptides. The summary of visualised neuropeptide by IMS is shown in Table 2. Nonetheless, as Chansela et al. report, using brain tissues previously prepared with paraffin-
Imaging by MALDI of paraffin-embedded sections of eyestalk, brain and thoracic ganglia tissues of shrimp Penaeus monodon. Composite Fig. 1A depicts a paraffin-embedded section (PETS) of P. monodon eyestalk tissue (upper left image) after hematoxylin–eosin (H&E) staining where the magnified structures of the retina (R), the lamina ganglionalis (LG), the medulla externa (ME), the medulla interna (MI) and the medulla terminalis (MT) can be seen. Under similar treatment conditions as above, the middle left image in Fig. 1A is a PETS of P. monodon brain tissue that shows the magnified structures of the olfactory lobe (OL) and the accessory neuropil (AL), and the lower left image is a PETS of P. monodon thoracic ganglia tissue with the magnified structure of the neuropil. The ion intensity maps of neuropeptides Phenylalanine-Methionine-Arginine-Phenylalanine (FMRF) and AYRKPPFNGSIF (SIF) amides can be seen Figs. 1B and 1C, which show that the strongest signals were detected in P. monodon thoracic ganglia and brain tissues, but especially in clusters 11 and 14 of the brain (white arrowheads). In contrast, at m/z 1338.6, crustacean hyperglycaemic hormone molecules (CHH) were found only in low levels in ME and MI of ES, the thoracic ganglia and clusters 11 and 14 of the brain (3D). Images and text adapted from Chansela et al., 2012, with permission.
© 2014 The Mass Spectrometry Society of Japan
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Fig. 2.
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Spectral and imaging data by MALDI MS of γ-aminobutyric acid, dopamine, and 3-methoxytyramine distributions in tissues sections of rat brain. (A) The composite spectrum generated by MALDI shows the individual peaks of γ-aminobutyric acid (GABA), catecholamine dopamine (DA) and 3-methoxytyramine (3-MT). After in situ derivatisation with 2,4-diphenyl-pyranylium tetrafluroborate (DPP-TFB), Shariatgorji et al. successfully visualised in sagittal sections of rat brain tissue (B), the relative abundance and distribution of GABA (C), DA (D), and 3-MT (E). GABA was found to be most abundant in the hypothalamus (HY) and the basal forebrain (BF), whereas DA (D) and 3-MT in the stantia nigra (SN), the caudate-putamen (CP), the ventral striatum (STRv) and the anterior olfactory nucleus (AON). Images and text adapted from Shariatgorji et al., 2014, with permission.
embedded tissue sections (PETS) greatly help to image neuropeptides by MALDI IMS, because histological structures are preserved and acetone or ethanol can be used to remove lipids by dehydration.37) Chansela et al. successfully visualised by MALDI IMS the distribution of CNS neuropeptides in PETS of shrimp Panaeusmonodon, with no reported analytical problems37) (Fig. 1). In a similar manner, after embedding brain tissues of crab Cancer borealis in paraffin blocks, Chen et al. used a 3D mapping technique to localise the distribution of several neuropeptides.38)
Imaging mass spectrometry techniques for amino acid neurotransmitters
Some amino acids can act as neurotransmitters by mediating the signalling between neurons, and between neuron and glia cells. Glutamate, γ-aminobutyric acid (GABA), and serine, amongst others, are considered amino acids capable of transmitting these signals.29) In the classic view, glutamate is the major mediator of excitatory signalling in the CNS,44) whereas GABA is an inhibitory neurotransmitter.45) Nonetheless, Chavas and Marty46) recently showed that a dual role (inhibition and excitation) of GABA was found in the cerebellar interneuron network. In contrast, serine is involved in long-term potentiation, fear conditioning and nociception, and abnormal levels of this amino acid have been linked to chronic neurodegenerative disease and Alzheimer’s disease.47) Although glutamate and GABA have been extensively analysed by chromatography techniques,48,49) IMS analysis of amino acid neurotransmitters is still an incipient area of research. Weak ionisation and interference by ion signals of matrices during IMS detection © 2014 The Mass Spectrometry Society of Japan
can be listed as the technical challenges that have prevented visualisation and quantification of amino acid neurotransmitters by IMS.5) With modifications to pre-column derivatisation and HPLC/ESI-tandem MS techniques previously reported by Nakatsukasa et al.,50) Toue at al. developed a derivatisation procedure for sections of murine tumourbearing liver tissues and analysed them by MALDI IMS to obtain semi-quantitative spatial information on several amino acids including glutamate, GABA and serine.5) Using an ingenious approach, Sugiura et al. combined IMS with focused microwave irradiation—which effectively stopped and preserved in vivo brain metabolism in brain tissues—to quantify and map glutamate in glutaminergic neuron-rich regions.51) Furthermore, very recently Shariatgorji et al. reported a promising technique for the direct and absolute quantitative imaging of neurotransmitters including GABA and glutamate, which addresses not only the interference by matrix signals but also the poor ionisation efficiency by MALDI52) (Fig. 2).
Imaging mass spectrometry techniques for small molecule neurotransmitters
Acetylcholine (ACh) was the first compound to be defined as neurotransmitter and its primary role is to act as an excitatory neurotransmitter in muscular synapses, except in the heart, where it inhibits signal transmission.25,29) Abnormal ACh signalling has been associated with Huntington’s, Alzheimer’s and Parkinson’s diseases, and schizophrenia.29) There are only few, but nevertheless critical technical impediments that complicate ACh analysis by IMS. For example, in a recent experiment, we underlined the fact Page 4 of 9
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Fig. 3.
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Tandem IMS analysis of acetylcholine in tissue sections of murine spinal cord. Our laboratory successfully imaged the spatial distribution of acetylcholine (ACh) in murine spinal cord tissues by conducting tandem IMS analysis. Figure 3A shows the average spectra of the total product ion data generated by IMS. ACh-derived product ion was detected at m/z 87 (inset), which was obtained as a neutral loss of a trimethyl-amine group (NL59) from the original intact ion at m/z 146. Figure 3B shows the intensity ion map after reconstruction and Fig. 3C depicts the final ion distribution map of galactosylceramide after merging. Arrowheads (3C) show cholinergic motor neuron-containing regions in the murine spinal cord where ACh was found to be most abundant. Images and text adapted from Sugiura et al., 2012, with permission.
that, since ACh exhibits a high degradation rate, an efficient sample preparation was needed to minimise post-mortem deterioration.14) To that end, we used liquid nitrogen to flash-freeze brains tissues of deeply anaesthetised mouse, a technique known as in situ freezing, which minimised postmortem degradation whilst improving dynamic range and sensitivity. We also found that interference by the matrix signals to that of the precursor ion was another problem. We successfully avoided this by detecting ion transitions in tandem MS14) (Fig. 3). The use of common matrices such as DHB and CHCA is yet another technical hindrance, as these compounds usually cause interference for ACh detection by IMS. Shariatgorji et al. avoided matrix interference during the MALDI analysis of ACh by using the deuterated form of CHCA (D4-CHCA). Matrix D4-CHCA overcomes interference by shifting signals by a minimum of 4 m/z units.53) In a different approach, Ye et al. tested the resolution capacity of MALDI LTQ Orbitrap XL MS, and found that it was powerful enough to separate analyte signals even from those of traditional matrices. Indeed, using MALDI LTQ Orbitrap XL MS, Ye et al. accurately mapped ACh in mouse brain tissues coated with DHB as matrix.54) Serotonin (5HT), dopamine (DA), norepinephrine (NE), © 2014 The Mass Spectrometry Society of Japan
and epinephrine (EP) are amines that act as neurotransmitters, of which DA is considered the most abundant amongst them.25) Previous data show that DA pathways are associated with perception of reward and mediation of learning and feeding, and thus addictions are strongly related to the dopamine system.25) Similarly, NE and EP are thought to be neurotransmitters with excitatory roles in brain functions related to arousal, attention, learning, memory and stress response.55) In contrast, 5HT regulates brain functions during alertness and motor activity as well as primal behaviours such as eating, sleeping and reproduction.56) Nevertheless, disorders of the 5HT systems have been associated with depression, anxiety, and schizophrenia.25) Imaging MS analysis of these amine transmitters has been limited by similar challenges as those faced by IMS amino acid analysis, although a few experiments have achieved certain success. For example, NE and EP levels were detected in the adrenal gland of adult pigs by IMS without matrix.57) Likewise, visualisation and quantitation of dopamine in brain tissue by MALDI IMS was possible by derivatising dopamine with 2,4-diphenyl pyrylium using CHCA as matrix.29) Purine ring-containing nucleosides adenosine, AMP, ADP, and ATP are signalling molecules of about 300– 1000 Da that have been found to act as neurotransmitters.29) Page 5 of 9
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Fig. 4.
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Imaging by MALDI IMS of in situ distribution of adenosine, adenosine monophosphate, adenosine diphosphate and adenosine triphosphate in control mice and ischemic rats. Images show in situ changes in abundance of adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) during ATP metabolism in coronally-sectioned brain tissues of control and ischemic rodent models, after being subjected for 24 h to middle cerebral artery occlusion. The adenosine structure was identified by comparing MS or tandem MS spectra with those of a standard compound. Scale bar: 1 mm. Images and text adapted from Liu et al., 2014, with permission. ISCH: tissue images of ischemic rats, CON: tissue images of control mice.
Adenosine and ATP are excitatory neurotransmitters in the nervous system that mediate signalling to motor neurons in the spinal cord and autonomic ganglia.25,29) As with most small molecules, attempts to measure and visualise purine neurotransmitters by IMS have been hindered by rapid degradation rates, low concentrations and matrix-related signal interference. In a ground-breaking work, Suematsu and colleagues successfully mapped the distribution of UDP,51) and AMP, ADP, and ATP58) in mouse brain tissues. To achieve this, they first minimised post-mortem degradation of tissue samples by either in situ freezing58) or irradiating them with focused microwave—which has been discussed above.51) Next these workers used stand-alone MALDI IMS,51) or MALDI IMS combined with CE-ESI-MS,58) to quantify and visualise metabolic fluxes of UDP-glucose complex, and to construct maps of adenine nucleotides for identification of AMP, ADP, and ATP in absolute terms (e.g., µM/g of brain tissue), respectively. In a more recent work, Liu et al. deposited 1,5-naphthalenediamine (1,5-DAN) hydrochloride onto the brain tissue sections of control and ischemic rodent models, using an automatic matrix sprayer, which permitted a homogeneous matrix coating of the whole tissue surface. Matrix 1,5-DAN showed strong ultraviolet absorption, high tolerance to salts and fewer background signals, especially in the low mass range (m/z 500 or lower).59) With this approach, Liu et al. were able to successfully map by MALDI IMS the distribution of adenosine, AMP, ADP, and ATP in the tissue sections, and estimate the changes of these metabolites during ATP metabolism58,59) (Table 1, Fig. 4).
© 2014 The Mass Spectrometry Society of Japan
SECONDARY ION MASS SPECTROMETRY, A COMPLEMENTARY TECHNIQUE TO MALDI Secondary ion mass spectrometry (SIMS) is a hard ionisation IMS technique that, unlike MALDI, targets elements, and small mass compounds (
