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Quantification of biogenic amines from individual GFPlabelled Drosophila cells by MALDI-TOF mass spectrometry. Max Diesner, and Susanne Neupert Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00961 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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Analytical Chemistry

Quantification of biogenic amines from individual GFP-labelled Drosophila cells by MALDI-TOF mass spectrometry Max Diesner, Susanne Neupert*

University of Cologne, Department of Biology, Institute for Zoology, Zülpicher Strasse 47b, 50674 Cologne, Germany

*Correspondence to: [email protected]; phone: +49 221 470 2867; University of Cologne, Department for Biology, Institute for Zoology, 50674 Cologne, Germany

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Analytical Chemistry 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 Cell-cell communication plays a crucial role in orchestrating and modulating neural circuits. To understand such interactions, it is vital to determine and quantify the involved messenger molecules such as neuropeptides and biogenic amines on the level of single cells. In this study, we used single-cell mass spectrometry (SCMS) to qualify and quantify octopamine (OA) and tyramine (TA) from isolated single cells from intact brains of the fruit fly Drosophila melanogaster. Our workflow involved targeted GFPguided

single-cell

microdissection,

on-plate

chemical

derivatization

with

4-hydroxy-3-

methoxycinnamaldehyde (CA) or 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde (DPD) for increasing ion stability and ion signal intensity, and isotopically marked internal standards for quantification by MALDITOF MS. We were able to determine a limit of detection for OA of 1 fmol/µl, for TA of 2.5 fmol/µl and a lower limit of quantification (LLOQ) of 10 fmol/µl for both substances. SCMS of GFP-labelled somata from ventral midline neurons of the labial neuromere (VMlb) of the gnathal ganglion revealed an OA titer of 17.38 fmol/µl and a TA titer (~2.5 fmol/µl) lower than the LLOQ, independent of sex. However, using a genetically altered driver line devoid of OA, TßhnM18/Tdc2>GFP, we confirmed TA in these cells. Furthermore, cold-anesthetization of flies caused a significant increase in OA content in VMlb somata. We compared OA titers of somata from two different OA/TA cell clusters to demonstrate the usefulness of targeted SCMS in advancing our understanding of OA/TA signaling in behavior and physiology. An influence on the detection of neuropeptides by our derivatized SCMS method could be excluded.

KEYWORDS: Drosophila single cell MALDI-TOF MS, octopamine, tyramine, quantification, 4hydroxy-3-methoxycinnamaldehyde (CA), 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde (DPD)

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Introduction Neuroactive substances such as biogenic amines and neuropeptides modulate many physiological processes and behavioral patterns in organisms. In the central nervous system (CNS), they are produced in specific neurons and can act as neurotransmitters, neuromodulators, and/or neurohormones. To investigate the functional effects of these substances in well-defined neural circuits, robust and highly sensitive measurement tools are necessary. Most notably, with a focus on the chemical characterization of minute volume samples, single-cell mass spectrometry (SCMS) has become an enabling technique for studying cellular molecule profiles, including neuroactive substances such as peptides, proteins, amino acids, and biogenic amines1,2. In order to study biogenic amines, high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) coupled with laser-induced native fluorescence (LINF) detection3 and electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS)4,5 are well-established approaches for identification and quantitation of these substances up to the level of single cells6. While direct neuropeptide profiling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) from single cells is a commonly used tool in neurobiology7-11, the analysis of biogenic amines using MALDI-TOF MS from individual cells is still an analytical challenge. Detection of these substances using MALDI-TOF MS is often hampered by the generation of intense matrix signals in the corresponding mass ranges of interest, in-source analyte instability, insufficient molecule ionization, or putative ion suppression events. To overcome these hurdles, chemical derivatization12-14, selection of matrix15,16, and matrix preparation17 have been assessed to increase ion sensitivity and analyte stability in MALDI-TOF MS. Furthermore, combining an optimized matrix preparation with internal standard application, quantification of small molecules from dried droplet samples has been successfully performed17-20. Neuroactive compounds, such as neuropeptides and biogenic amines, however, can be colocalized in specific neurons, but an analytical tool to identify and map single neurons regarding aminergic compositions within an intact brain is still lacking. In this study, we fill this gap by developing a biogenic amine identification and quantification protocol on SCMS, focusing on cells of the octopaminergic/tyraminergic system. We used the fruit fly Drosophila melanogaster as model system, as it offers the possibility to use promoter-specific GAL4 drivers to target specific cells or cell populations in the CNS. In Drosophila and other invertebrates, the biogenic amine octopamine (OA), equivalent to norepinephrine in vertebrates, is synthesized in a two-step process; tyrosine is decarboxylated by tyrosine decarboxylase-2 (Tdc2) into tyramine (TA)21, which is followed by the hydroxylation of TA into OA by tyramine-β-hydroxylase22,23. The functions and localizations of OA and TA in Drosophila have been studied extensively, mainly using genetic labeling tools and immunohistochemistry21,24-27, showing that OA is involved in controlling behaviors such as aggression28,29 and sleep30, modulating sensory systems such as taste31 and vision32, as well as higher brain functions such as learning and memory33. In contrast, 3



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only little is known about the function of TA, with recent studies implicating an involvement in courtship34, flight35, and olfaction36. From an analytical point of view, the first studies quantifying biogenic amines such as OA and TA from different developmental stages of individual Drosophila brains have been conducted37,38. However, such experimental setups have the drawback of sample homogenization, which renders identification and quantification of biogenic amines from a single cell as well as unambiguous cell localization in an intact brain impossible. Here, we used fluorescence-guided microdissection in conjunction with a tyrosine decarboxylase-2 (Tdc2)-GAL4 line crossed to a UAS-mCD8::GFP reporter line for identification of TA and OA-containing cells in intact brains. We tested two commercially available derivatization agents for enhancing sensitivity and stability of OA and TA ions and investigated detection and quantification limits by combining on-plate derivatization with isotopically labelled internal standards. Furthermore, we compared the concentrations of OA and TA from individual isolated cells from a specific cell population between females and males, investigated temperature-dependent effects on OA/TA concentrations, and determined OA/TA titer differences in two cell populations at the single-cell level. To demonstrate the application of our approach also for neuropeptide detection, we analyzed single peptidergic Drosophila neurons.

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Experimental section Chemicals: Chemicals were purchased from Sigma-Aldrich unless stated otherwise.

Synthetic solutions: Stock solutions of OA hydrochlorid, TA hydrochlorid, (±)-p-octopamine-α,β,β-d3 hydrochloride (OA[d3]), CDN Isotopes, Pointe-Claire, Canada) and 2-(4-hydroxyphenyl)ethyl-1,1,2,2-d4amine hydrochlorid (TA[d4]; CDN Isotopes) were prepared in 50% MeOH/TraceSELECT® water at a concentration of 10 nmol/µl, stored at 4°C in darkness and were used to prepare dilution series. Stock solutions of synthetic biogenic amines were replaced after two months, dilution series after three weeks. Derivatization reagents: Based on Manier et al.14, 4-hydroxy-3-methoxycinnamaldehyde (CA) was prepared in 100% MeOH at a concentration of 23 mg/ml and centrifuged at 13000 rpm at 4°C for 10 min. 5 µl of the supernatant was diluted in 150 µl 50% MeOH/TraceSELECT® water at a concentration of 0.76 mg/ml. According to Gatti et al.39, 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde (DPD) was dissolved in 50% MeOH/TraceSELECT® water at a concentration of 0.415 mg/ml. The mixture was vortexed for 30 s and sonicated for 2 min in ice water. Both solutions were centrifuged at 15000 rpm for 10 min at 4°C for use and prepared fresh daily.

Fly stocks: Adults flies ≥ 5 days old of both sexes were used. Flies were raised on standard cornmeal, molasses, yeast, agar medium on a 12 h/12 h light-dark cycle at 25°C and 60% humidity. The following fly strains were used: Tdc2-GAL421; UAS-mCD8-gfp33; TßhnM18-GAL42 (kindly provided by Manuela Ruppert, University Cologne); c929-Gal440 (kindly provided by Christian Wegener, University Würzburg); TßhnM18,FM7; TdC2-GAL4, TdC2-GAL4 (TßhnM18/ TdC2-GAL4, this work).

Quantification and standard curve calculation: Sample preparation for analysis of OA/TA was modified from Persike et al.18. To calculate standard curves, either 300 nl (18.4 nl) of synthetic OA, TA, or a mixture of both was applied onto a MALDI sample plate in a concentration range from 1000 to 0.1 fmol/µl diluted in 50% MeOH/TraceSELECT® water. For pMS² standard curves, only samples between 100 and 1 fmol/µl were analyzed. The spots were air-dried and covered with 300 nl (18.4 nl) OA(d3), TA(d4), or a mixture of both with a constant concentration of 100 fmol/µl. After air-drying again, either 126 nl (9.2 nl) of CA or DPD were added onto the spots. Then, each sample spot was covered with 300 nl (18.4 nl) α-cyano-4-hydroxycinnamic acid (CHCA) and dried under constant airflow. CHCA was prepared fresh daily in 80% MeOH/TraceSELECT® water at a concentration of 1.43 mg/ml. Volumes < 100 nl were applied using a nanoliter injector (NL2000, World Precision Instruments, Berlin, Germany). 5



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Single-cell dissection: Flies were either immobilized for 60 min or 15 min on ice, or directly removed from the vials using forceps. The brain was removed in dissection buffer (NaCl 126mM, KCl 5.4mM, NaH2PO4 0.17mM, KH2PO4 0.22mM, pH 7.4) under an epifluorescence stereomicroscope. For single-cell dissections, brain samples were transferred to a fresh drop of ice cold dissection buffer containing 33% glycerol. The ganglionic sheath around the area of a GFP-labeled cell was removed; the soma manually picked using an uncoated glass capillary and transferred onto a MALDI target plate. Excessive dissection saline was removed using the same glass capillary. Residual glycerol surrounding the isolated soma was washed off with 50% MeOH/TraceSELECT® water. Washing was performed with a fresh glass capillary until the glycerol was completely removed; however, the soma itself was left uncovered to prevent loss of analytes. In general, only one cell body was isolated per brain sample, and the complete dissection did not exceed 1 h.

Quantification of OA and TA from single cells: For sample quantification, 18.4 nl of OA(d3) and TA(d4) at a concentration of 100 fmol/µl were added to each sample, followed by 9.2 nl of CA or DPD and 18.4 nl of CHCA. Each sample spot was dried under a constant stream of air. Between each application step, remaining solution in the glass capillary was removed and rinsed two-times with 100% TraceSELECT® water, 50% MeOH/TraceSELECT® water followed by 100% MeOH before air-drying.

MALDI-TOF mass spectrometry: Mass spectra were acquired using an UltrafleXtreme MALDITOF/TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). All acquisitions were performed under manual control in reflector positive ion mode using an 2kHz Bruker smartbeam-IITM laser. The instrument settings were optimized for the mass ranges of m/z 0-400 and m/z 600-4000, respectively. The instrument was calibrated using prominent CHCA matrix ion signals17 for the mass range m/z 0-400 and using synthetic peptides for the mass range at m/z 600-4000. All mass spectra were acquired with 2000 laser shots with a laser frequency of 333 Hz and a laser focus diameter at ~70 µm (medium).

MS/MS experiments: MS/MS experiments were performed using LIFT technology, with an acceleration of 1 kV. The number of laser shots varied from 1000 to 2000 for biogenic amines and from 2000 to 5000 for neuropeptides, depending on ion signal quality. To verify OA and OA(d3), MS/MS spectra were acquired without collision gas. For the validation of TA and TA(d4), fragmentations were performed in collision-induced dissociation (CID) mode with argon as the collision gas. Resulting fragments of underivatized substances were compared to data provided in the Scripps Center for Metabolomics database (METLIN, https://metlin.scripps.edu/index.php). Peptide identities were verified by comparison 6


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of predicted (http://prospector.ucsf.edu) and experimentally obtained fragment ions. For quantification experiments of OA and TA, mass spectra were acquired in parent ion mode with fixed laser intensity. The ion window was set from m/z -0.4 to +6. Each mass spectrum was acquired with 2000 laser shots. A signal was considered to be detected with a minimum S/N ratio of 3. The data obtained in these experiments were processed with the FlexAnalysis 3.4 software package (Bruker Daltonik GmbH, Bremen, Germany). For quantitative analysis, only unprocessed data were used.

Method validation and statistics: Method validation of standard curves followed the ±15/20 criteria published by the US Food and Drug Administration (FDA)41. All data points for standard curve calculation are averages of at least three replicates. Linear ranges, accuracy, relative standard deviations (RSD) and linearity (R²) were calculated with Microsoft Excel 2010 and/or R 3.1.3 (R Development Core Team). To compare OA titers between different octopaminergic cell populations, data points were tested for normal distribution using a one-sample Kolmogorov-Smirnov test and were either analyzed by student’s t-test or one-way analysis of variance (ANOVA) for datasets with more than two groups. Imaging: For imaging of native GFP expression, brains were handled as described by Pitman et al.42. Brains were dissected in ice-cold phosphate-buffered saline (PBS, 1.86 mM NaH2PO4, 8.41 mM Na2HPO4, 175 mM NaCl) and fixed in 4% paraformaldehyde diluted in PBS for 90 min at room temperature (~21° C) in vacuum (300 mbar). Then, samples were washed three times for 10 mins in PBS containing 0.1% Triton X-100 (PBT) and one time in PBS before mounting in 90% glycerol/1% DABCO/9% PBS. Imaging was performed on a Zeiss LSM Meta 510 microscope (Zeiss AG, Jena, Germany), and resulting images were processed in Amira 5.4.2 (FEI, Hillsboro, OR). Reconstruction of brain surfaces was performed manually in Amira, while GFP-marked cells were visualized using voltex rendering.

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Results and Discussion This study demonstrates a workflow to qualify and quantify the neurotransmitters octopamine (OA) and tyramine (TA) from targeted isolated single cells up to 7 µm from intact Drosophila brains.

Analysis of non-derivatized and derivatized synthetic octopamine (OA) and tyramine (TA): Synthetic non-derivatized OA and TA covered with CHCA were analyzed to evaluate the analytical platform in terms of OA and TA ion stability and limits of detection (LOD). The resulting mass spectra revealed that OA and TA were both unstable during measurements and underwent fragmentation during gas phase transition (OA, m/z 154.1 intact, m/z 136.0 water loss43; TA, m/z 138.1 intact, m/z 121.0 amino group loss43; Figure 1A1, 1B1, S1). Subsequent MS² experiments of OA and TA ions showed the same ion fragments observed in the MS spectra. Due to the observed molecule instability and poor ionization, we were not able to detect OA at levels lower than 100 fmol/µl and TA at levels lower than 10 pmol/µl, even in MS² mode. For an unequivocal identification of OA and TA from biological samples as small as a single Drosophila cell, ion stability had to be notably increased. In the recent years, a number of derivatization reagents and protocols for neurotransmitter detection using MALDI-TOF MS were developed to improve ion stability, specificity, and sensitivity, mostly for molecules with a primary amine group12-16,39. We tested two of these derivatization agents: 4hydroxy-3-methoxycinnamaldehyde (CA)14 (Figure 1A2, 1B2) and 2,5-dimethyl-1H-pyrrole-3,4dicarbaldehyde (DPD)39 (Figure 1A3, 1B3). An exemplary reaction scheme of CA and DPD with OA is shown in Figure 1C. All CA-derivatized samples showed a mass shift of m/z +160 in resulting mass spectra (Figure 1A2, 1B2), as previously reported14. Thus, OA-CA is detectable in MS mode as m/z 314.1 (Figure 1A2), and TA-CA as m/z 298.1 (Figure 1B2). MS² experiments of OA-CA revealed a distinct ion signal at m/z 296.1, which represents the derivatized ion fragment of m/z 136.0 from non-derivatized OA (Figure 1A2, S1,2). Resulting MS² mass spectra of derivatized TA showed a clear fragmentation pattern consisting of three distinct ion signals at m/z 120.8, 178.0, and 243.0 (Figure 1B2, S1,2) using CID. For identification of TA-CA, we compared latter MS2 data with data of a baseline ion signal at m/z 298.1 from samples containing only CA, which demonstrated that only the ion fragment at m/z 120.8 could be used to unambiguously identify TA-CA. In terms of ion stability and sensitivity, we observed, for both OA-CA and TA-CA, a striking increase that resulted in LODs of 25 fmol/µl for OA (4-fold decrease) and 5 fmol/µl for TA (5000-fold) in MS mode. Furthermore, we determined the LODs of OA-CA and TA-CA in MS² mode after derivatization and again observed decreased LODs for OA-CA of 1 fmol/µl (100-fold) and for TA-CA of 2.5 fmol/µl (7500-fold; see Table 1). For analysis with DPD, the resulting mass spectra revealed a mass shift of m/z +133 for both OA (m/z 154.1 → m/z 287.1; Figure 1A3, S3) and TA (m/z 138.1 → m/z 271.1; Figure 1 B3, S3). Subsequent structure evaluation in MS² experiments yielded three distinct ion signals at m/z 136.1, 152.1, and 269.1 8


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for OA-DPD (Figure 1A3, S3) and ion signals at m/z 121.1, 136.1, and 151.1 for TA-DPD (Figure 1B3, S3), in accordance with previous findings obtained by ESI-MS experiments39. To identify OA-DPD and TA-DPD using MS², we analyzed samples containing only DPD covered with CHCA. The resulting MS2 mass spectra recorded from baseline ion signals at m/z 287.1 and m/z 271.1 revealed that only the ion signals at m/z 152.1 for OA-DPD and m/z 121.1 for TA-DPD represent unique fragment ions for reliable identification. The determined LODs for OA-DPD and TA-DPD show a value of 10 fmol/µl for both substances in MS mode and of 1 fmol/µl in MS² mode (see Table 1). The achieved LODs for OA and TA with both derivatization agents were satisfying; however, for SCMS, our derivatization approach had to be scaled to much smaller volumes. Therefore, we tested and optimized our derivatization protocol with 18.4 nl samples which confirmed our determined LODs for OA and TA from larger-volume samples (Table 1).

Detection of OA and TA from individual Drosophila somata: For reliable cell identification, we used a Tdc2-GAL4 driver line crossed to a UAS-mCD8::GFP reporter line (Figure 2A1, 2A2). All neurons labeled by Tdc2>GFP should contain TA, and, according to earlier studies, most of these neurons also contain OA26,27. To test the effectiveness of our method on the single-cell level, we focused on ventral midline (VM) neurons located in the labial neuromere (VMlb) of the gnathal ganglion (GNG). These easily accessible cells showed a sufficient GFP signal in almost all Tdc2>GFP fly brain samples, which is essential for manual single-cell dissection under a fluorescence stereomicroscope. For initial single-cell experiments, somata were prepared in dissection buffer containing 33% glycerol to stabilize neuron morphology and reduce the release of neuroactive substances from the soma into the axon without changing the biochemical profiles of cells44,45. Only cells without any visible debris and an intact fluorescence-labeled cell soma on the sample plate were analyzed (Figure 2A2). MS analysis of single VMlb cells derivatized with CA revealed an ion signal at m/z 314.1 (Figure 2 B1) which was confirmed as OA-CA (Figure 2B2). Recorded mass spectra from DPD-derivatized VMlb cells contained an ion signal at m/z 287.1 (Figure 2C1), which was confirmed as OA-DPD by subsequent fragmentation experiments (Figure 2C2). TA measurement after CA and DPD derivatization was not convincingly verified with recorded mass spectra although a minor ion signal in a few samples, identical with derivatized TA, was found and subsequent MS² experiments confirmed the ion as TA-CA or TA-DPD (Figure S5). To prove if these findings are based on a very low titer of TA in VMlb cells and originate not from the derivatization, we analyzed single VMlb somata from the OA-devoid transgenic line TßhnM18/Tdc2>GFP. This driver line shows elevated TA titers resulting from the absence of tyramine-ßhydroxlase, the enzyme converting TA to OA23,46. Mass spectra from single TßhnM18/Tdc2>GFP VMlb somata treated with CA yielded clear ion signals at m/z 298.1, corresponding to putative TA-CA (Figure 2D1). Subsequent fragmentation revealed a clear identification of TA-CA (Figure 2 D2). Thus, the 9



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fluctuating detection of TA from Tdc2>GFP VMlb somata was due to a very low TA titer in these cells, which is at the boundary of the LOD (~2.5 fmol/µl) of our developed protocol.

Isomeric ion species and neuropeptide profiling: To exclude potential isomeric ion species originating from samples, as well as to test the influence of the derivatization agents on neuropeptide detection, we analyzed individual peptidergic drosomyosuppressin (dms)-expressing neurons labeled by the c929>GFP diver line (Figure 3A)11, which do not belong to the octopaminergic/tyraminergic system of D. melanogaster26,27. Resulting mass spectra after CA treatment revealed two ion signals at m/z 1247.6 and m/z 1407.5 (Figure 3B, S6). Resulting MS² spectra confirmed the presence of DMS and DMS-CA, as compared to synthetic test results (Figure 3C, S6). For potential isomeric ion species, we analyzed the same sample spot in a mass range of m/z 0-400. Recorded mass spectra showed no additional ion signals in MS mode or in MS² experiments (Figure 3D-F, S6). Similar results were found for DPD derivatization experiments (Figure S7). Thus, our approach can be utilized to detect biogenic amines and neuropeptides using SCMS. Futthermore, putative interference from dopamine (DA), which is mass identical to OA, was excluded by tandem mass experiments of DA-CA (m/z 314.1) and DA-DPD (m/z 287.1; Figure S4). Both fragmentations revealed unique ion pattern, differing from OA-CA and OA-DPD MS2 experiments (Figure S4).

Quantification of synthetic OA and TA: To construct standard curves for quantification, sample spots of 300 nl of OA and TA in a concentration range of 1000 fmol/µl to 0.5 fmol/µl followed by 300 nl of 100 fmol/µl of internal standard comprised of OA(d3) or TA(d4), 126 nl CA or DPD, and 300 nl of CHCA were prepared. For analysis, each concentration was measured with at least three replicates and the average ion intensity ratios were calculated. Obtained standard curves for CA revealed robust RSDs (< 15/20%), good precision (< 15/20%), and excellent linearity values in MS mode (OA-CA, R² = 0.9997; TA-CA, R² = 0.9999), which fulfilled the criteria given by the FDA41 (Figure S8). Our experimentally determined lower limits of quantification (LLOQs) for OA-CA and TA-CA were 25 fmol/µl and 5 fmol/µl, respectively. The quantification of small molecules using MALDI-TOF MS, however, can often be hampered by interference from matrix ion signals. To circumvent this issue, we also determined the LLOQs for both substances using the parent MS2 (pMS²) mode (Figure S8), which provides the option to detect ions of interest in a selected mass range and exclude thereby neighboring interfering ion signals. For that, we defined the ion window for OA-CA and TA-CA quantification from m/z -0.4 to +6, which resulted in a lower LLOQ at 2.5 fmol/µl for OA-CA (Figure S3) and a constant value of 5 fmol/µl for TACA (Table 1). pMS² standard curves were only analyzed in the range from 100 fmol/µl to 1 fmol/µl. Next, we determined the LLOQ for smaller samples with 18.4 nl internal standard as previously described. Determined standard curves for OA-CA revealed the same LLOQ at 25 fmol/µl for OA-CA in 10


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MS mode (Table 1), while, in pMS² mode, the LLOQ was set to 10 fmol/µl (Figure 4A, Table 1, Table S1) due to day-to-day variation, possibly resulting from varying matrix interference. Data acquired in MS and pMS2 mode for TA-CA confirmed previously observed LLOQ values from 300-nl sample spots (Figure 4B, Table 1, Table S1). Due to the small-volume sample spot as well as equal LLOQ values for TA-CA in MS and pMS2 mode, all further TA-CA experiments were performed in MS mode. A summary of determined LOQ values of OA and TA measurements using different analytical approaches is presented in Figure S9. Experiments with DPD, however, resulted in RSD values exceeding the 15% limit, poor precision, and/or declined linearity, conceivably due to a slower reaction time leading to a putative incomplete turnover and, therefore, not qualifying for a robust quantification in conjunction with our protocol (Figure S10). Finally, we assessed the stability of samples stored in light and dark conditions. Samples were stable for over 69 h when stored in the dark, while samples stored in the light were only stable up to 6 h (Figure S11).

Quantification of OA and TA in VMlb somata: In the next step, we applied our optimized quantification protocol to single Tdc2>GFP-labeled VMlb neurons. In order to achieve reproducible results, all measurements were recorded in the following order: (1) pMS2 mode for OA-CA quantification, (2) MS mode for TA-CA quantification and fingerprinting, (3) MS² mode for OA-CA and OA(d3)-CA confirmation, and (4) MS² mode TA-CA and TA(d4)-CA confirmation. First, VMlb somata quantification experiments of animals cooled for 60 min on ice were prepared, and recorded mass spectra showed clear ion signals for OA-CA with an average concentration of 29.67 fmol/µl (n = 14, ± 7.49 fmol/µl). Recorded TA-CA titers, however, were below the LLOQ, but tandem MS experiments revealed evidence for TA-CA in 3 of 14 samples. This supports our previous finding that somata in the VMlb cluster contain very low concentrations of TA but a relatively high titer of OA.

Temperature-dependent variability of detectable OA titers in VMlb neurons: 47,48

behavioral experiments often rely on animal immobilization by cooling

Physiological and

, however, the influence of this

intervention on the physiology of the organism remains largely unexplored. To answer these questions, we again prepared VMlb somata from two additional test groups: (1) uncooled animals (t = 0) and (2) animals cooled for 15 min (t = 15). Recorded mass spectra showed an average OA-CA concentration of 17.38 fmol/µl (n = 17, ± 1.94 fmol/µl) for 15-min-cooled samples and 13.67 fmol/µl (n = 21, ± 3.25 fmol/µl) for uncooled samples. Statistical comparisons of all three sample groups (uncooled, 15 min, 60 min) revealed significant differences in OA titers (ANOVA, uncooled ~ 15 min, p = 0.02; uncooled ~ 60 min, p = 8.59e14; 15 min ~ 60 min, p = 1.01e-9) (Figure 5A), demonstrating a direct correlation between duration of 11



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cooling and detectable neurotransmitter concentration and possibly mediated by a reduced neurotransmitter vesicle transport from the cell soma to the axon with the biosynthesis process still intact. In all samples recorded, TA-CA titers were below the LLOQ, but tandem MS revealed clear evidence for TA-CA in 13 of 21 cells (male, 7 of 11; female, 6 of 10) for uncooled samples and 10 of 17 cell samples (male, 7 of 9; female 3 of 8) after 15 min of cooling.

Sexual dimorphism: Behavioral and physiological changes associated with OA signaling are crucial for reproductive success, mating, and aggression in Drosophila. For instance, OA signaling in females is involved in modulation of postmating responses49 whereas, in males, OA is a key player in the regulation of fighting behavior28,29. To test if sexual dimorphism is reflected in the variation of OA-CA concentration in VMlb cells, we compared VMlb somata from uncooled (Figure 5B) and 15-min-cooled (Figure 5C) female and male flies. Recorded mass spectrometric data for females showed average OA-CA concentrations of 12.65 fmol/µl (n = 10, ± 2.04 fmol/µl) for uncooled animals and 17.37 fmol/µl (n = 8, ±1.79 fmol/µl) after 15 min of cooling, which was comparable to those of male flies with an average OACA value of 14.59 fmol/µl (n = 11 ± 3.70 fmol/µl) for uncooled animals and 17.40 fmol/µl (n = 9, ± 1.96 fmol/µl) after 15 min of cooling. Statistical comparison of OA titers between sexes revealed no sexual dimorphism for both sample groups (uncooled, t-test, p = 0.17; 15 min cooled, t-test, p = 0.97) (Figure 5B,C).

Quantification of OA from two OA/TA cell populations: Finally, we compared OA-CA concentrations of single somata from two different octopaminergic/tyraminergic cell populations: (1) cells from the analyzed VMlb cluster and (2) somata from the ventrolateral cluster (VL). (Figure 2A1,2) The VL cluster is characterized by two neurons per hemisphere with different target regions in the CNS26. It has been shown that VL neurons modulate bitter gustatory receptor neuron output via a potential co-release of OA and TA31. However, it is still not completely clear whether these neurons produce OA, as immunocytochemical stainings against tyramine-β-hydroxylase showed no labeling of these cells28, while other studies showed immunofluorescence targeting OA and TA in this cluster26. To address this issue, we analyzed VL neurons from male and female flies that were cooled for 15 min on ice. Mass spectra from VL neurons revealed ion signals for both OA-CA and TA-CA. MS² experiments confirmed the presence of OA-CA (Figure 6A) in all samples and the presence of TA-CA (Figure 6B) in 5 out of 9 samples. An explanation for this phenomenon could be a time-dependent expression of Tβh, resulting in a discontinuous OA processing with TA still present. Furthermore, our findings are supported by the fact that the target cells of VL neurons express Oct-TyrR, a receptor that reacts to OA and TA to modulate bitter taste31. Thus, considering our findings, we could clearly show that these neurons synthesize both OA and TA as potential neuroactive transmitters. 12


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Even though concentrations of OA-CA in VL somata were below the LLOQ based on FDA criteria, we calculated OA-CA values from recorded pMS² spectra of analyzed VL neurons with an average concentration of 3.98 fmol/µl (n = 9, ± 2.03 fmol/µl) to render it possible to compare VMlb and VL OA-CA titers statistically. Comparison of recorded VMlb and VL OA-CA titers showed a highly significant difference between the two cell clusters (Figure 6C; t-test, p = 1.19e-10), highlighting that somata concentrations of neurotransmitters such as OA can vary tremendously between cell clusters with different functions, whereas intra-cluster variation seems to be low.

Conclusion The ability to qualify and quantify neurotransmitters such as OA and TA from small-volume samples down to the level of single cells in Drosophila offers a potential new measurement tool for characterizing cell-to-cell heterogeneity, physiological processes, neural dysfunction, and behaviorassociated changes in organisms. This approach is highly sensitive, reproducible, cost-effective, fast, and adaptable with slight optimizations regarding analytes of interest, soma size, and cell types. Furthermore, our on-plate derivatization protocol for neurotransmitter quantification can be combined with intracellular recordings for characterization of cell properties, followed by dye injection for neuroanatomical evaluation, before SCMS analysis. The aim of future studies should be to develop tools to analyze neurotransmitter levels at axon endings and synapses within intact tissues to monitor release processes of specific compounds under different physiological, pharmaceutical, and environmental conditions within a neurologically relevant context.

Supporting Information MS² spectra of un/derivatized deuterized standards, DA and suggested chemical structure of detected fragments; Detection of derivatized TA in VMlb samples; Characterization of synthetic and cellular DMSCA/DPD; linearity, precision and accuracy of 300 nl CA/DPD and 18.4 nl OA/TA-CA samples; Comparison of LOQs; analysis of sample degradation during storage.

Acknowledgments We thank financial assistance of the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG; SN: NE911/3-1).

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Table 1. Summary of experimentally determined LODs and LLOQs for OA-CA and TA-CA analysis in MS, MS², and pMS² mode. (*) = 300-nl samples; (**) = 18.4-nl samples.

calculated values LOD OA-CA TA-CA OA-DPD TA-DPD LLOQ OA-CA TA-CA

MS [fmol/µl]

MS2/pMS² [fmol/µl]

25 5

1 2.5

10 10

1 1

25*/25** 5*/10**

2.5*/10** 5*/10**

16


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Figure 1: Mass spectrometric analysis of underivated, CA-, or DPD-derivatized synthetic OA and TA. Representative MS and MS² spectra of (A) synthetic OA and (B) TA (1) without derivatization reagents and (2) after CA and (3) DPD treatment. (A1, B1) Both underivated OA (m/z 154.1) and TA (m/z 138.1) are unstable represented by ion fragments at m/z 136.1 (OA) and m/z 121.0 (TA) in MS mode, which was confirmed by subsequent MS² experiments. (A2, B2) Derivatization with CA (m/z +160) led to increased ion stability and ion signal intensity of OA (A2: OA-CA, m/z 314.1) and TA (B2: TA-CA, m/z 298.1). MS² experiments of derivated substances showed that OA-CA is identified by the ion fragment m/z 269.1 and TA by m/z 120.8. (A3, B3) Derivatization of OA and TA using DPD (m/z +133) and subsequent fragmentation analysis for ion confirmation. (C) Chemical reaction scheme of CA and DPD with OA. Arrows: ion fragments for identification.

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Figure 2: Analysis of OA and TA from derivatized single Drosophila somata using direct MALDI-TOF MS. (A) Anterior (1) and posterior (2) views of brain of Tdc2>GFP driver line. VM, ventral midline; VL, ventrolateral cells; VMmx, VM maxillary cluster; VMmd, VM mandibular; VMlb labial cluster. Scale bar, 25 µm. Inset: Isolated single GFP-labeled soma on the sample plate. (B) Detection (1) and confirmation (2) of OA from individual dissected somata after (B1,2) CA and (C1,2) DPD derivatization. (D1) Mass spectra of a single TβhnM18/Tdc2>GFP VMlb somata to evaluate the efficiency of our protocol for TA identification on single cell level and (D2) TA confirmation by MS2. Arrows: ion fragments for identification.

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Figure 3: Effects of sample derivatization with CA and DPD on neuropeptide profiling using drosomyosuppressin (dms)-expressing cells. (A) Posterior view of a c929>GFP brain sample. Right circle: DMS cells are marked. Left circle: Single DMS cell was isolated and placed onto a MALDI sample plate (insert). Scale bar, 25 µm. (B) Mass spectrum of derivated single DMS cell revealed two ion signals at m/z 1247.6 and m/z 1407.5. (C) MS² spectrum of m/z 1407.6 which confirmed DMS-CA. (D) MS spectrum of DMS cell in a mass range of m/z 0-440. (E,F) MS² analysis of background ion signals showed (E) no OA and (F) no TA.

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Figure 4: Standard curves constructed from 18.4-nl volume samples for quantification of (A) OA-CA in pMS2 and (B) TA-CA in MS mode with observed linearity and precision.

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Figure 5: Statistical analysis of temperature-dependent OA titers recorded from VMlb cells. (A) Cooling resulted in distinct increases of OA titers. Significant differences were found between all three VMlb sample groups. (ANOVA, uncooled x 15-min cooling, p = 0.02; uncooled x 60-min cooling, p = 8.59e14; 15 min~60 min, p = 1.01e-9). No sexual dimorphism was observed for (B) uncooled (t-test, p = 0.17) or (C) 15-min-cooled (t-test, p = 0.97) flies.

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Figure 6: Detection of OA-CA and TA-CA in VL neurons and comparison of OA-CA concentrations from VMlb and VL cells. (A) MS² spectrum of putative OA-CA from a single VL neuron. The ion identity was confirmed as OA-CA by the ion fragment m/z 296.1. (B) Confirmation of TA-CA from VL samples by tandem MS by detection of m/z 120.7. (C) Statistical comparison of OA titers from VMlb and VL samples revealed a highly significant difference between two cell populations (t-test, p = 1.19e-10).

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For TOC only

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