Enhanced Coverage of Insect Neuropeptides in Tissue Sections by an

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Enhanced Coverage of Insect Neuropeptides in Tissue Sections by an Optimized Mass Spectrometry Imaging Protocol Alice Ly, Lapo Ragionieri, Sander Liessem, Michael Becker, Sören Oliver Deininger, Susanne Neupert, and Reinhard Predel Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04304 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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Analytical Chemistry

Enhanced Coverage of Insect Neuropeptides in Tissue Sections by an Optimized Mass Spectrometry Imaging Protocol Alice Ly1‡*, Lapo Ragionieri2‡*, Sander Liessem2, Michael Becker1†, Sören-Oliver Deininger1, Susanne Neupert2*, Reinhard Predel2* 1Bruker

Daltonik GmbH, Fahrenheitstraße 4, 28359 Bremen, Germany for Biology, Institute of Zoology, University of Cologne, 50674 Cologne, Germany

2Department

ABSTRACT: Mass spectrometry imaging (MSI) of neuropeptides has become a well-established method with the ability to combine spatially resolved information of immunohistochemistry with peptidomics information of mass spectrometric analysis. Several studies have conducted MSI of insect neural tissues, however; these studies did not detect a neuropeptide complement comparable to that obtained with conventional peptidomics. The aim of our study was to improve sample preparation so that MSI provides comprehensive and reproducible neuropeptidomics information. Using the cockroach retrocerebral complex, the presented protocol produces enhanced coverage of neuropeptides at 15 µm spatial resolution, confirmed by parallel analysis of tissue extracts using electrospray ionization MS. Altogether, more than 100 peptide signals from 15 neuropeptide precursor genes could be traced with high spatial resolution. In addition, MSI spectra confirmed differential prohormone processing and distinct neuropeptide-based compartmentalization of the retrocerebral complex. We believe that our workflow facilitates incorporation of MSI in neurosciencerelated topics, including study of complex neuropeptide interactions within the CNS. Keywords: MALDI-MS imaging, spatial segmentation analysis, neuropeptidome, differential prohormone processing, retrocerebral complex, insect nervous system INTRODUCTION: Neuropeptides are structurally diverse signaling molecules which control and regulate essential physiological functions in vertebrates and invertebrates, including growth, feeding, reproduction, and environmental stress tolerance. A major source of neuropeptides is the central nervous system (CNS) where neuropeptides can act as transmitters or neuromodulators. Alternatively, neuropeptides can be produced in neurosecretory cells within the CNS and released as peptide hormones into circulation; mostly from neurohemal organs which function as a hormone repository. The large number of neuropeptides and neuropeptide receptors generally hampers decoding of coordinated peptide actions. Some neuropeptide precursors may result in mature peptides that activate different receptors; e.g. melanocyte stimulating hormone precursors of vertebrates and CAPA precursors of insects,1-3 which further complicates the full recognition of neuropeptide actions. Mass spectrometry is increasingly used to analyze the neuropeptidome of the CNS even to single-cell level.4,5 Although the aim of many of these approaches is to decipher neuropeptide relationships or compensation strategies, a number of limitations persist in the study of such complex neuropeptide interactions. In insects, which include notable model organisms in neuropeptide research such as the fruit fly Drosophila melanogaster and honeybee Apis mellifera, small tissue size, low peptide abundance and complex cellular pattern of peptidergic neurons still necessitate the extensive use of immunohistochemistry (IHC) to complement neuropeptidomic studies. IHC has traditionally been used to investigate neuropeptide distributions in the CNS of insects but has limited ability for visualizing neuropeptides from different precursors in the same sample, even when using

fluorochrome-coupled secondary antisera. Usually, IHC also fails to discriminate between sequence-related precursor products. This problem is evident in insects for numerous RFamides which are both processed from single precursors (up to 20 extended FMRFamides) and encoded by additional genes such as myosuppressin, sulfakinin, short neuropeptide F, and long neuropeptide F. In this context, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) could be an ideal alternative technique for studying the spatial distribution of neuropeptides in the nervous system.6-8 MALDI-MSI been successfully employed for analyzing brain peptides of crustaceans.9,10 Few studies have so far used MALDI-MSI for detection of insect neuropeptides and the reported tissue preparations and/or spatial resolutions (30 µm and greater) were usually not sufficient to discriminate small structures within the insect nervous system.11,12 A study using prototype MSI instrumentation examined lipid distributions in 20 µm D. melanogaster whole body sections with pixel sizes ranging between 5-10 µm, and also reported several mass matches with neuropeptides.13 In all of these studies, however, mass spectra did not detect neuropeptide contents comparable to that obtained with analysis of single dissected neurons or direct tissue profiling.5,14 We investigated the suitability of MALDI-MSI for the analysis of neuropeptide distributions in the retrocerebral complex (RCC) of the American cockroach, Periplaneta americana, a model organism in neuropeptide research, and developed an optimized MSI protocol to analyze as much of the neuropeptidome as possible. The RCC is the major neuroendocrine organ in insects comparable to the pituitary gland of vertebrates. A recent MALDI-MSI study was able to detect several neuropeptides in 30 year old paraffin-embedded

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RCC samples.15 In comparison with insect brains whose complexity hampers an easy recognition of specific areas by MSI, the organization of the RCC is easier to reconstruct and facilitates reproducibility.

Figure 1. Overview of the P. americana RCC (dorsal view) and its junctions with brain and stomatogastric nervous system (SNS). The black line indicates the area of the brain from which come the nerves that supply the RCC with neurosecretion. Neurosecretory cells in the pars intercerebralis and the pars lateralis of the protocerebrum are indicated by green and blue circles, respectively. Dotted lines represent the respective pathways leading to the nervi corporis cardiaci. NCC, nervus corporis cardiaci; NCA, nervus corporis allati; NCS, nervus cardiostomatogastricus; SEG, subesophageal ganglion.

Figure 1 is an overview of the P. americana RCC and its connections to key neurological structures. The RCC consists of a pair of corpora cardiaca which is fused posteriorly to a pair of corpora allata. While the corpora allata synthesize sesquiterpenoids (juvenile hormones), the corpora cardiaca exclusively release peptide hormones. Each corpus cardiacum is subdivided into a glandular part antero-dorsally, producing insect's equivalent of glucagon, the adipokinetic hormones (AKHs)16 and the remaining parts of the corpora cardiaca which serve as neurohemal release site of numerous peptide hormones from the brain and subesophageal ganglion (SEG). These hormones reach the RCC via different nervi corporis cardiaci (NCC) and nervi corporis allati-2 (NCA-2).17,18 Axons from NCA-2 as well as a number of axons from neurosecretory cells of the brain cross the corpora allata and contribute to neuropeptide detection along these glands. The RCC is connected to the stomatogastric nervous system (SNS) by way of the nervi cardiostomatogastrici (NCS). The products of a large number of neuropeptide genes of insects can be found in the RCC,18-21 but the exact neuropeptide-based compartmentalization of the RCC is still largely unknown. Our data demonstrate the extent to which MALDI-MSI with commercially available instrumentation can be used to reconstruct the distribution of neuropeptides in an insect nervous system. With the described protocol, we obtained good coverage of the neuropeptides expected to be present in the RCC. In addition, the spatial distribution of further neuropeptides could be verified for the first time in the RCC. EXPERIMENTAL SECTION

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Chemicals and Reagents. Alpha-cyano-4hydroxycinnamic acid (CHCA), and peptide calibration standard II were purchased from Bruker Daltonik GmbH (Bremen, Germany). HPLC-grade ethanol and acetonitrile were obtained from Honeywell (Seelze, Germany). Trifluoroacetic acid (TFA) was purchased from Merck (Darmstadt, Germany). Standard food-grade gelatin purchased from local supermarkets (Dr. Oetker Gelatin white, Bielefeld, Germany) was used in this study. An ELGA Purelab flex system (Veolia; Celle, Germany) was used to generate deionized water. Animal model and Sample Preparation. The animals in this study were treated pursuant to the Declaration of Helsinki. Cockroaches were raised and maintained at a constant temperature (28 ± 1 °C) under a 12 h light/dark cycle with free access to food and water. For experiments, adult cockroaches were kept at 4 °C for 30 min before RCCs were dissected in insect saline (NaCl 126 mM, KCl 5.4 mM, NaH2PO4 0.17 mM, KH2PO4 0.22 mM; pH 7.4), rinsed in deionized water, and embedded in 100 mg/ml gelatin/water. The gelatin was dissolved in deionized water, heated to 80 °C for five minutes to ensure dissolution, and then kept at 50 °C to maintain viscosity. For tissue embedding, 400 µl of dissolved gelatin were poured into handmade aluminum foil molds with 8 mm internal diameter and allowed to solidify at room temperature for at least 30 min. Subsequently, the RCCs were horizontally placed on the solidified gelatin and then slowly covered with 200 µl of gelatin at approximately 30 °C. The embedded tissue was snap-frozen at -50 °C immediately after. RCC samples were cryosectioned (-10 °C) at 14 or 20 µm thickness with a 10-degree blade angle on a Microm 550 cryostat (Thermo Fisher; Walldorf, Germany), and thaw-mounted onto indium tin oxide (ITO) coated glass slides (Bruker Daltonik). Finally, the samples were stored at -80 °C until MSI measurement. Prior to matrix application, the samples were removed from the freezer, brought to room temperature, and dried in a nitrogen-rich environment using an ImagePrep device (Bruker Daltonik) and stored under vacuum (300 mbar). Following this, samples were either not washed at all or washed at room temperature in 70 % v/v ethanol/water and 100 % ethanol for 20 seconds each with an interval of five seconds drying time between each wash. The latter probes were dried again under vacuum (300 mbar) for 1 h at room temperature. After optical scanning (TissueScout; Bruker Daltonik), the sections were coated with 5 mg/ml CHCA dissolved in different ratios of acetonitrile/water/TFA using a SunCollect Dispenser System (SunChrom, Friedrichsdorf, Germany). Matrix was sonicated and filtered before being sprayed. SunChrom spray control software v2.5 was used to deposit eight layers of matrix using variable spray rates (10 µl/min, 20 µl/min, 30 µl/min, 40 µl/min, 50 µl/min; three layers at 60 µl/min) at a speed of 900 mm/min. The line distance (Y-direction) was set to 2 mm and spray nozzle height (Z-position) was 25 mm. Immunohistochemistry. Samples were fixed in 4 % paraformaldehyde diluted in phosphate buffer saline (PBS; pH 7.2) at 4 °C for 30 min and subsequently washed three times in PBS for 30 min. The samples were pre-incubated with 5 % normal goat serum dissolved in PBS for 30 min, then incubated for 12 h at 4 °C in rabbit anti-P. americana corazonin serum (1:4000; kindly provided by J. Veenstra) and mouse anti-Diploptera punctata allatostatin A-7 serum (1:200; 5F10 kindly provided by B. Stay) diluted in PBS, respectively. Following washing (3 x 30 min), samples were incubated with goat anti-mouse Cy2- (1:500) and goat-anti-rabbit Cy3-


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Analytical Chemistry (1:3000) tagged secondary antibodies (Jackson Immuno Research, West Grove, PA, USA) at 4 °C for 12 h. Finally, samples were mounted in entellan and stored at 4 °C. Image processing. Immunostainings were examined with a confocal laser scanning microscope (ZEISS LSM 510 Meta system; Jena, Germany), equipped with an Apochromat 10x/0.45W (NA 0.45) objective using the multi-track mode. Cy2 was excited at 492 nm and emission collected by a BP 505-530 filter and Cy3 was excited at 543 nm and emission collected via a LP 560 BP filter. Serial optical sections were analyzed with 0.9 μm thickness each and assembled into combined images using the Zeiss LSM 5 image browser version 3. The final figures were exported and processed to adjust brightness and contrast with Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA). Preparation of RCC extracts. Extracts of P. americana RCC were prepared as described.22 Briefly, a single P. americana RCC was extracted in 20 μl of solution containing 50 % methanol/water and 1 % formic acid. Extracts were sonicated for a few seconds then centrifuged for 15 min at 13 000 rpm. Supernatants were transferred to fresh sample tubes (0.5 ml) and either used for Quadrupole Orbitrap MS or MALDI-TOF MSI. For MSI experiments, 0.3 µl of supernatant was deposited on an ITO glass slide and allowed to dry. Matrix was applied using the SunCollect sprayer as described for imaging experiments. This procedure was repeated three times to reduce batch effects and the results compared. The extract was measured as a single spectrum using the MSI acquisition parameters as an imaged area (average 50 pixels/area). MALDI Mass Spectrometry Imaging. MALDI-MSI was performed using a rapifleX MALDI-TOF Tissuetyper mass spectrometer (Bruker Daltonik) in positive ion reflector mode over a mass range of m/z 600-3200, with a 15 µm laser spot size and 15 µm lateral step size. For each measurement position, 500 laser shots were accumulated using a Smartbeam 3D Nd:YAG (355 nm) at a frequency of 5000 Hz and sample rate of 1.25 GS/s; with baseline subtraction (TopHat) during acquisition. The instrument was calibrated using peptide calibration standard II spotted onto the matrix-coated ITO glass slide, taking care that the spot was not obscuring the tissue. Ion images were generated using flexImaging v. 5.0 and SCiLS Lab MVS software version 2018a (Bruker Daltonik) with data normalized to the Total Ion Count (TIC). Reduced data (Bruker .dat files) were uploaded and preprocessed for a time-of-flight (TOF) instrument in SCiLS, and underwent spatial segmentation analysis using a bisecting kmeans with correlation distance approach.23 The default pipeline was used with the following modifications: medium denoising and ± 0.30 Da interval width. 10 RCC preparations with the corresponding sections (3-5 sections of each RCC preparation) were analyzed using the MSI protocol. Quadrupole Orbitrap Mass Spectrometry. RCC extract was analyzed with a Q-Exactive Plus (Thermo Fisher Scientific, Waltham, MA, USA) as described.24 Prior to injection, the sample was desalted using self-packed Stage Tip C18 spin columns. Using PEAKS 8.5 (PEAKS Studio, BSI, Canada) and MaxQuant (v. 1.6.2.10, MPI, Martinsried, Germany), MS2 spectra were compared against an internal database containing known P. americana neuropeptides14 and a newly annotated Neuropeptide like Precursors 1 (NPLP1 – Acc. Nr. MH837510). For both pipelines, the maximum number of PTMs (sulfation [Tyr], C-terminal amidation,

Cystine, oxidation [Met, Trp], pyroglutamyl formation [Glu, Gln], N-terminal acetylation [Lys]) per peptide was set at five and no digestion mode was selected. For analyses using MaxQuant, the first search peptide tolerance was set at 20 ppm and main search peptide tolerance was set at 4.5 ppm. The False Discovery Rate (FDR) was set to 0.01 for peptide spectrum match, and only peptides with a P-score > 60 were considered for manual inspection. For peptide search using PEAKS, the parent error mass tolerance was set at 10 ppm, a fragment mass error tolerance at 0.05 Da the FDR below 1 % and fragment spectra with a peptide score (−10 lgP) equivalent to a P-value of about 1 % were selected and manually reviewed. Statistics. Paired t-tests were used to calculate the effect of different method parameters (GraphPad Prism (v. 5.04), San Diego, CA, USA). RESULTS AND DISCUSSION Conceptualization of an imaging protocol for insect neuropeptide analysis. To yield as much neuropeptidomic information as possible, we tested different approaches for sample preparation utilized throughout the MALDI-MSI field (for a review see Buchberger et al.).7 As a guide, we used the neuropeptide complement from a RCC extract detected with the same setup for sample preparation (including matrix sprayer, matrix composition, matrix application procedure) and MALDI-TOF (same mass analyzer, ionization technique, sample target device, instrument settings) that were used for imaging experiments. No significant degradations of peptides in any of our experiments were found, an obvious advantage of using insect tissue samples. Analysis of the tissue extracts revealed 60 mature neuropeptides which could be assigned to 15 neuropeptide precursor genes of P. americana (Table S1). Assignment of ion signals to neuropeptides of P. americana was supported by MS2 data from Quadrupole Orbitrap analyses of a RCC extract (Figure S1). For our first MSI experiments with RCC sections, we adapted a protocol for MALDI imaging of the honeybee brain,11 but with a matrix sprayer for deposition of CHCA instead of a matrix spotter. This approach revealed peptidomic information which was much less comprehensive than that obtained from RCC extracts. In fact, only few abundant peptides were detected with weak spatial distribution (data not shown). Therefore, we re-evaluated each experimental step to reach an optimized MSI protocol suitable for a comprehensive analysis of neuropeptides in RCC tissue sections (see also Figure S2). Step 1 – Dissection: To avoid excessive release of peptides during dissection, we used cold saline solution during preparation of the RCC. Before embedding, the isolated RCC was washed in ice-cold deionized water for a few seconds to remove saline solution and to avoid salt crystal formation during embedding-freezing steps. As a general rule, short dissection times of less than 5 min and the strict avoidance of direct contact of RCC tissue with the forceps resulted in more consistent mass spectra along the complete tissue sections. For the transfer, we used the nerves that leave the RCC towards the periphery. Step 2 – Embedding: The small size of the RCC (approximately 0.5 x 1 mm) made it necessary to embed the tissue prior to sectioning. Gelatin is an embedding substrate known to be compatible with MALDI-MSI.9,25 Chen et al. used 100 mg/ml gelatin in water for MALDI-MSI of crustacean brain neuropeptides;9 this concentration also


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worked with the much smaller RCC tissue. We also tested gelatin concentrations ranging between 80 to 120 mg/ml without obtaining better results; sectioning quality was decreased with lower gelatin concentration, while the increased density of more concentrated gelatin reduced the ability to properly embed the samples. Accurate horizontal placement of the RCC, which consists of two mirror-imaged parts, facilitated quality control by comparing peptide distribution in the two parts. We found that an optimal positioning was achieved by using two layers of gelatin. The RCC was placed and oriented on the solid lower layer; any remaining water was carefully removed around the RCC by using a glass capillary before the sample was slowly covered with the more fluid (warmer) gelatin. Step 3 – Cryosectioning: MSI of whole RCC has previously been reported,12 but thicker tissues are not highly electrically conductive which can result in poor spectra.26 In addition, only peptides located at the outer margin of the RCC are likely to be analyzed when performing whole tissue profiling combined with matrix spraying. In order to obtain uniform tissue sections with reproducible mass spectrometry profiles, tests were performed using different section thicknesses (5 - 20 µm), cutting temperatures (-20 °C to -10 °C) and blade cutting angle (5 – 20 °). The best section quality without folding or squeezing tissue sections was achieved with a blade angle of 10 °, temperature at -10 °C and with a tissue thickness of 1420 µm; while cutting thinner sections was possible, it is more difficult to maintain tissue integrity. Tissue sections were serially collected on ITO glass slides using manual control of cutting pace and stored at -80 °C. For optimal peptide coverage in mass spectra, the samples were dried under vacuum at about 300 mbar for at least 12 h after defrosting. Shorter drying times (e.g. 1 h) decreased the peptide coverage significantly (p = 0.0006, Figure S3). Step 4 – Ethanol washes of tissue sections: Neuropeptide analysis by MSI from crustacean neuronal tissues was reported without washing for whole tissue or sections prior to matrix application,9 MSI of neuropeptides from whole Drosophila sections was performed without washing the tissue sections but the complete animals were immersed in ethanol prior to sectioning.13 The use of ethanol washes has been reported to remove lipids and salts that can interfere with peptide and protein signals.11,27 We observed statistically significant lower neuropeptide coverage and strong interference from lipid species when washing was omitted (ttest, p = 0.0004, Figure S4). Two consecutive ethanol washes using first 70 % v/v ethanol/water followed by absolute ethanol for 20 seconds each, provided the best coverage of neuropeptides in the samples (Figure S4). After washing, the sections were dried again for at least 1 h at 300 mbar to ensure removal of ethanol. The ethanol concentrations used in our MSI experiments have been reported for detection of intact proteins28 and slightly modified from the detection of A. mellifera brain neuropeptides.11,29 For the comparison of peptide coverage we selected regions of interest (ROI; 200 x 200 µm) within CA tissue which showed more uniform peptidome in consecutive sections compared to other parts of the RCC. The average number of peptides identified within the ROIs was significantly higher for washed samples (10.63 ± 1.133; N = 8) than for samples prepared without washing steps (4.625 ± 0.7055; N = 8). The good neuropeptide coverage in mass spectra obtained from washed samples was accompanied by a slight decrease of resolution in MSI ion maps. For those peptides which were detectable in sections

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without washing (e.g. pyrokinins), we therefore used both approaches in parallel. The obtained differences in resolution indicate a certain degree of delocalization of peptides during the washing. Therefore, further decreasing the laser spot size or the size of matrix crystals might have little effects in these samples. Step 5 – Matrix application: We used CHCA which is a matrix preferred for the detection of peptides and small proteins,30 and has been reported for detecting neuropeptides in MSI experiments on honeybee brains11,29 and flatworms31. For high-spatial resolution measurements, smaller matrix crystal sizes are desirable as large crystals can lead to analyte spread and require more energy for ionization.32 The spraying device employed in this study has previously been used for detecting small molecules,33,34 N-glycans, tryptic peptides,34 and insect neuropeptides.11,29 The chosen parameters (spray rate, spray head speed, spray head distance from sample, number of cycles) were selected based on a combination of visual inspection of matrix deposition (size, even distribution, no convergence of droplets) during the cycles and analysis of peptide yields in subsequent mass spectrometry experiments. For example, when altering spray speeds and rates, we ensured that the sprayed layers were completely dry before starting the next layer and comparing with how many neuropeptide signals were detected. In order to ensure reproducible results, an ITO glass slide was always coated with matrix to test the functionality of the sprayer. For that, the glass slide was weighed, coated with matrix and weighed again to estimate the amount of deposited matrix. Coating of samples commenced if the matrix evenly covered the slide and weighed between 0.9 - 1.0 mg. The initial matrix composition, 5 mg/ml CHCA dissolved in 70 % ACN/H2O with 0.1 % TFA, was successively modified to 5 mg/ml in 50 % ACN/H2O with 2 % TFA. Increasing TFA concentrations resulted in higher signal intensity of neuropeptides in MSI spectra and a significant increase of peptide coverage (p = 0.0167, Figure S5). Using these spraying conditions we obtained matrix crystals of about 20 µm, which corresponds roughly to the 15 µm laser spot size used in our analyses. Smaller laser spot sizes (5 - 10 µm) were tested but failed to generate a full peptidome in subsequent mass spectra. High-Spatial Resolution MALDI-MSI of Multiple Copy Peptides in the RCC. A number of mature insect neuropeptides are processed as multiple copies (paracopies) from precursor proteins. These paracopies are usually processed in equimolar ratios. Among the known cockroach neuropeptides present in the RCC, allatostatin A (AstA) peptides, extended FMRFamides (FMRFs), myoinhibitory peptides (MIPs), sulfakinins (SKs), kinins, and pyrokinins (PKs) have several paracopies ranging in number from two (SKs) to 21 (FMRFs).14 These paracopies ideally show (1) constant relative signal intensity among each other in MSI spectra and (2) identical spatial distributions. Hence, analysis of paracopies in MSI spectra provides information regarding spectra quality. MSI ion maps from a single section consistently verified a similar distribution of the different FMRF paracopies which all have specific sequences in P. americana (Figure 2A). The presence of FMRF ion signals was consistent in mass spectra of MSI tissue sections (Figure 2B) and extract samples that have been spotted on ITO glass slides before being analysed with the same MALDI-TOF equipment (Figure 2C). Analysis of all sections of single RCCs also revealed the overall distribution of these peptides along the RCC. It has to be noted


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Analytical Chemistry that the distribution of FMRFs in the RCC was resolved although these peptides showed low signal intensities in mass spectra from RCC extracts (Figure 2B). Differential Distribution of Neuropeptides in the RCC/ SNS. Subsequent to the confirmation that MSI spectra show a reliable spatial distribution of neuropeptide paracopies, we analyzed the distribution of all mature neuropeptides detected in our MSI spectra. Altogether, we observed ion signals of 57 mature neuropeptides, a number that matches well with the number of neuropeptide ion signals obtained by extract analysis (Table S1). The number of observed peptides exceeded 100 if additional precursor peptides (cleavage products without known functions) were included.

Figure 2. FMRF paracopies in mass spectra from RCC preparations. (A) MSI from a single tissue section showing distribution of four FMRFs suggesting identical spatial distribution of these peptides in the RCC. 20 µm section; scale bar: 200 µm; Ion intensity bar: 100-20 %. (B) Mass spectrum obtained by means of MSI; the analyzed spot is indicated in (A) by an arrow. (C) Mass spectrum obtained by means of “MSI” of an aliquot of a RCC extract spotted on an ITO glass slide. Matrix spraying and MALDI-TOF equipment exactly as used for (B). Accuracy of mass matching for peptide assignment was settled at ± 0.25 Da.

Figure 3 exemplarily shows neuropeptide distributions in two RCC sections. The local accumulation of peptides from various neuropeptide genes within the RCC differed dramatically and recalled some old neuroanatomical studies which described distinct axonal pathways within the seemingly uniform RCC.35,36 The ion maps only partially matched with the few available immunostainings depicting the distribution of neuropeptides in the RCC but corresponds with data obtained by direct tissue profiling of parts of the RCC and SNS.37 A brief summary of the distribution pattern of neuropeptides in the RCC/SNS as revealed by MSI is given hereinafter. Myosuppressin and sk genes are both expressed in neurosecretory cells of the pars intercerebralis in the protocerebrum with projection into the RCC via the NCC-1. The neuropeptides from these genes showed a distinct accumulation within the RCC (Figure 3A and 3B) that is different from each other and from those of the other RFamide peptides such as short neuropeptide F (Figure 3C) and FMRFs (Figure 3F). For kinins and MIPs which were found by IHC in cells of the pars lateralis and pars intercerebralis,18,38 we also observed different distribution patterns. Kinin accumulation was restricted to the corpora cardiaca while MIPs were more abundant along the SNS (Figure 3D and 3E). As exemplarily shown for PK-3, PKs were most abundant around the corpora allata (Figure 3G); a detailed description of the distribution of PKs is given in the following section on prohormone processing. The two AKH peptides, which are products of different genes, were restricted to the glandular antero-dorsal part of the RCC (Figure 3J). The only peptide entirely restricted to the SNS was proctolin (Figure 3K), whereas CCAP was observed in the neurohemal partof the corpora cardiaca only (Figure 3 L).

Figure 3. MALDI-MSI ion maps confirming the differential distribution within the RCC/SNS of neuropeptides from 12 different genes. (A) Pea-SK, m/z 1443.6 ± 0.25 Da, ion intensity bar: 100-20 % (B) Myosuppressin [pQ], m/z 1257.6 ± 0.25 Da, ion intensity bar: 100-20 % (C) Short neuropeptide F, m/z 1315.7 ± 0.25 Da, ion intensity bar: 100-20 % (D) Kinin-1, m/z 949.5 ± 0.25 Da, ion intensity bar: 100-40 % (E) MIP-2, m/z 1389.6 ± 0.25 Da, ion intensity bar: 100-35 % (F) FMRF-15, m/z 1159.6 ± 0.25 Da, ion intensity bar: 100-20 % (G) PK-3, m/z 996.6 ± 0.25 Da, ion intensity bar: 100-20 % (H) NPLP-1, m/z 1585.8 ± 0.25 Da, ion intensity bar: 100-20 % (I) Allatotropin, m/z 1366.7 ± 0.25 Da, ion intensity bar: 100-20 % (J) AKH-1, m/z 973.5 ± 0.20 Da, ion intensity bar: 100-10 % (K) Proctolin, m/z 649.4 ± 0.25 Da, ion intensity bar: 100-20 % (L) CCAP, m/z 956.5 ± 0.25 Da, ion intensity bar: 100-40 % (see Figure 1 for overview of the


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architecture of RCC/SNS). Scale bar (white): 600 µm. Section thickness (A-I): 20 µm, (J-L): 14 µm.

We also observed spatial distribution of neuropeptides not previously described by mass spectrometry in the RCC of P. americana, such as allatotropin (AT; Figure 3I, Figure S6), and FMRFs (see above, Figure 2). AT showed a distribution different from all other neuropeptides. Prominent ion signals were detected both in the SNS and in the corpora cardiaca, but rarely in the corpora allata (Figure 3I). It is unknown how AT enters the RCC, but based on the MSI information it seems possible that AT reaches the corpora cardiaca through the NCC-1. This assumption was substantiated by direct peptide profiling of isolated NCC-1 using conventional MALDI-TOF mass spectrometry (Figure S7). In addition to known cockroach peptides, multiple products of the neuropeptide-like precursor 1 (NPLP1), not reported in P. americana so far, could be identified. Peptides from NPLP1 precursors have been found in the CNS and RCC in several insects.39-41 The mass signals of 13 NPLP1 peptides were detected with distribution mostly restricted to the neurohemal part of corpora cardiaca (Figure 3H, Table S1, Figure S1).

Figure 4. Distribution of corazonin and AstA analysed in serial RCC sections by (A) immunohistochemistry and (B) MSI (the more peripheral section). Data obtained by both methods confirmed the different spatial distribution of corazonin and AstA, which are produced in cells of the pars lateralis of the brain, along the RCC. Labeling on the RCC margin is likely due to autofluorescence (detached gelatin). Scale bar: 200 µm; section thickness: 20 µm. Ion intensity bar: 100-20 %. Accuracy of mass matching for peptide assignment was settled at ± 0.25 Da.

Corazonin and AstA peptides, both of which are detected using IHC in cells of the pars lateralis of the protocerebrum with projection via the NCC-2 into the RCC 42,43 showed different distribution patterns. Corazonin signals were highly abundant in the neurohemal part of the corpora cardiaca, the nervus cardiostomatogastricus and adjacent parts of the SNS (Figure S6) but mostly not detectable along the corpora allata. This was the other way around with AstA signals, which were weak in mass spectra of the neurohemal part of the corpora cardiaca but in most preparations distinct around the corpora allata and the posteriorly directed part of the SNS (nervus esophageus). These differences were not expected according to IHC analyses. We therefore performed AstA/corazonin IHC double staining on peripheral RCC sections with less complex axon pathways and compared the staining patterns with MSI images from consecutive sections (Figure 4). Resulting data confirmed that AstA and corazonin indeed have a different spatial distribution along the RCC (see also Figure S6).

Figure 5. (A) Ion maps of four PKs indicating differential processing of the PK precursor. (B) Four PKs were detected in the posterior part of the RCC which mostly contains the PKs processed in cells of the SEG. (C) In contrast, the anterior corpus cardiacum tissue, which receives neuropeptides from the brain, does not show PK-1 ion signals. Section thickness: 20 µm; Scale bar: 200 µm; ion intensity bar 100-20 %, except for m/z 883.5: 100-35 %. Accuracy of mass matching for peptide assignment were settled at ± 0.25 Da for PK-2,3,4 and ± 0.001 Da for PK-1, respectively. Tissue section not washed with ethanol prior to matrix spraying.

Differential prohormone processing. An advantage of MSI is the capability to detect differential prohormone processing. In P. americana, differential processing is only demonstrated for the PK precursor. Whereas the full set of PKs is processed in cell clusters of the SEG with projection to the RCC via the NCA-2, few cells in the brain with projections into the RCC via NCC-1 do not process PK-1 and mass spectra of the NCC-1 therefore did not show ion signals of this PK.44 MSI spectra from the RCC confirmed these data (Figure 5). In the anterior corpora cardiaca near the junction with NCC-1 all PKs except PK1 were detectable, while all PKs were prominent in the neurohemal part of the RCC near the entrance of the NCA-2 and in the corpora allata. Two of the PKs (PK-1 and PK-3) have ion signals masssimilar to sodium and potassium adduct ions of AKHs. In MALDI-TOF mass spectrometry, AKHs are represented only by these adducts but ion maps verified, that even a mass difference of only 0.2 Da was sufficient to discriminate between these peptides (Figure S8). A previous MSI study of whole RCC tissue have shown similar ion signal discrimination.12 Once the accuracy of our MSI analyses was confirmed, we tested a bioinformatic approach based on the information obtained in MSI experiments. Application of spatial segmentation analysis to a single RCC section enabled the assignment of main compartments within the RCC/SNS (Figure 6). These regions correspond to the corpora allata and adjoining nervi corporis allati 1, corpora cardiaca glandular area, corpora cardiaca neurohemal area, and SNS. This result demonstrates statistical discrimination among the different areas independent from any a priori knowledge. Interestingly, the neurohemal area within the corpora cardiaca is further differentiated in three subcompartments; an anterior part surrounding the glandular tissue of the corpora cardiaca, a posterior portion with nervi cardiostomatogastrici and adjoining tissue, and the portion of the corpora cardiaca located between these parts. The respective dendrogram shows that the latter two compartments are more closely related to each other than to the neuroglandular area or SNS.


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Analytical Chemistry (AstA-11), corazonin (Crz) and allatotropin (AT) in consecutive RCC sections; Figure S7. MALDI TOF direct tissue profiling of a dissected nervus corporis cardiaci 1; Figure S8. Discrimination between mass-similar neuropeptides.

AUTHOR INFORMATION Corresponding Authors Figure 6. Spatial segmentation analysis of MSI data from a single RCC section. Different levels in the segmentation dendrogram represent distinct regions of the RCC corresponding to the corpora allata (CA) and nervi corporis allati 1 (NCA-1), glandular and neurohemal corpora cardiaca (CC); the neurohemal part of the corpora cardiaca is further subdivided in three subcompartments.

CONCLUSIONS This study provides a MSI workflow for analysis of neuropeptides in insect neuroendocrine tissues which results in a comprehensive neuropeptidome with high reproducibility, ion signal quality, and spatial resolution. Seemingly minor changes of established protocols produced an overall view of neuropeptide distributions with high-spatial resolution using conventional MALDI-TOF mass spectrometry equipment. Novelties included the distinct accumulation of different neuropeptides in the RCC/SNS which even holds for neuropeptides produced in different cell populations within a cell cluster. MSI experiments can potentially be incorporated in neuroscience-related topics such as complex changes in the neuropeptidome of insects which might be associated with development or adaptations induced by environmental stress, e.g. xenobiotics. The presented sample preparation protocol can certainly be used for other MALDI-MSI instrumentation, including those with higher mass and/or spatial resolution (e.g. MALDI-FT-ICR, AP-SMALDI-Orbitrap). Enhanced lateral resolution may be possible particularly in combination with spraying devices that result in smaller matrix crystal size or sublimation/re-extraction procedures.45-47 Commonly used washing steps prior to matrix application potentially result in analyte spreading and therefore might neutralize attempts to obtain a better lateral resolution. For RCC tissue, alternative tests without washes are rewarding; given that the peptide of interest is detectable with spatial resolution. If peptidomics information needs to include more extensive neuropeptide complement, washing steps are indispensable. In this context, the experiments presented in this study may serve as a guide when starting with other tissue preparations.

ASSOCIATED CONTENT Supporting Information: Table S1. List of mature neuropeptides from 15 precursor genes of P. americana; Figure S1. Quadrupole Orbitrap MS2 spectra of P. americana neuropeptides; Figure S2. Workflow for MSI sample preparation optimized for insect neuroendocrine tissue (RCC); Figure S3. Comparison of peptide coverage in tissue sections dried for a single h or 12 h before washing; Figure S4. Comparison of peptide coverage in tissue sections with and without successive ethanol washes; Figure S5. Comparison of peptide coverage in tissue sections after matrix spraying (5 mg/ml CHCA in 50 % ACN/H2O); with matrix solution containing 0.1 % TFA and 2 % of TFA; Figure S6. Distribution of allatostatinA-11

* Email: [email protected], Phone: +49-221-470-5817; [email protected], Phone: +49-221-470-8267; [email protected], Phone:+49-421-22054782; [email protected], Phone: +49-221-470-8592

Present Address †Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany

Author Contributions ‡These authors contributed equally. All authors contributed to the writing and have given approval to the final version of the manuscript.

Notes AL and SOD were employees of Bruker Daltonik GmbH for the duration of this study. MB was an employee of Bruker for part of this study. ORCID: Alice Ly: 0000-0002-0827-6840 Lapo Ragionieri: 0000-0003-0099-2719 Sander Liessem: 0000-0002-7073-2659 Susanne Neupert: 0000-0003-1562-5743 Reinhard Predel: 0000-0002-0202-6672

ACKNOWLEDGMENT This project was supported by the European Commission Horizon2020 Research and Innovation Grant 634361 (nEUROSTRESSPEP), German Research Foundation (PR 766/11-1), Graduate School for Biological Sciences Cologne (DFG-RTG 1960: Neural Circuit Analysis of the Cellular and Subcellular Level). We thank Susanne Hecht (Bruker Daltonik GmbH) for help in sample preparation.

REFERENCES (1) Cazzamali, G.; Torp, M.; Hauser, F.; Williamson, M.; Grimmelikhuijzen, C. J. P. The Drosophila Gene CG9918 Codes for a Pyrokinin-1 Receptor. Biochem. Biophys. Res. Commun. 2005, 335, 14– 19. (2) Iversen, A.; Cazzamali, G.; Williamson, M.; Hauser, F.; Grimmelikhuijzen, C. J. P. Molecular Cloning and Functional Expression of a Drosophila Receptor for the Neuropeptides Capa-1 and -2. Biochem. Biophys. Res. Commun. 2002, 299, 628–633. (3) Smith, M. E. POMC Opioid Peptides. In Handbook of Biologically Active Peptides; Kastin, A. J., Ed.; Elsevier, San Diego, 2006, pp 1325– 1331. (4) Rubakhin, S. S.; Sweedler, J. V. Characterizing Peptides in Individual Mammalian Cells Using Mass Spectrometry. Nat. Protoc. 2007, 2, 198797. (5) Neupert, S.; Johard, H. A. D.; Nässel, D. R.; Predel, R. Single-Cell Peptidomics of Drosophila melanogaster Neurons Identified by Gal4Driven Fluorescence. Anal. Chem. 2007, 79, 3690–3694. (6) Chen, R.; Li, L. Mass Spectral Imaging and Profiling of Neuropeptides at the Organ and Cellular Domains. Anal. Bioanal. Chem. 2010, 397, 3185-3193. (7) Buchberger, A. R.; DeLaney, K.; Johnson, J.; Li, L. Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights. Anal. Chem. 2018, 90, 240–265. (8) Spengler, B. Mass Spectrometry Imaging of Biomolecular Information. Anal. Chem. 2015, 87, 64–82. (9) Chen, R.; Cape, S. S.; Sturm, R. M.; Li, L. Mass Spectrometric Imaging of Neuropeptides in Decapod Crustacean Neuronal Tissues.


7


Methods Mol. Biol. 2010, 656, 451-463. (10) Chen, R.; Jiang, X.; Prieto Conaway, M. C.; Mohtashemi, I.; Hui, L.; Viner, R.; Li, L. Mass Spectral Analysis of Neuropeptide Expression and Distribution in the Nervous System of the Lobster Homarus americanus. J. Proteome Res. 2010, 9, 818–832. (11) Pratavieira, M.; da Silva Menegasso, A. R.; Garcia, A. M. C.; dos Santos, D. S.; Gomes, P. C.; Malaspina, O.; Palma, M. S. MALDI Imaging Analysis of Neuropeptides in the Africanized Honeybee (Apis mellifera) Brain: Effect of Ontogeny. J. Proteome Res. 2014, 13, 3054– 3064. (12) Verhaert, P. D. E. M.; Pinkse, M. W. H.; Strupat, K.; Conaway, M. C. P. Imaging of Similar Mass Neuropeptides in Neuronal Tissue by Enhanced Resolution MALDI MS with an Ion Trap - Orbitrap Hybrid Instrument. Methods Mol. Biol. 2010, 656, 433-449. (13) Khalil, S. M.; Pretzel, J.; Becker, K.; Spengler, B. High-Resolution AP-SMALDI Mass Spectrometry Imaging of Drosophila melanogaster. Int. J. Mass Spectrom. 2017, 416, 1-19. (14) Neupert, S.; Fusca, D.; Schachtner, J.; Kloppenburg, P.; Predel, R. Toward a Single-Cell-Based Analysis of Neuropeptide Expression in Periplaneta americana Antennal Lobe Neurons. J. Comp. Neurol. 2011, 520, 694–716. (15) Paine, M. R. L.; Ellis, S. R.; Maloney, D.; Heeren, R. M. A.; Verhaert, P. D. E. M. Digestion-Free Analysis of Peptides from 30-YearOld Formalin-Fixed, Paraffin-Embedded Tissue by Mass Spectrometry Imaging. Anal. Chem. 2018, 90, 9272–9280. (16) Gäde, G.; Hoffmann, K. H.; Spring, J. H. Hormonal Regulation in Insects: Facts, Gaps, and Future Directions. Physiol. Rev. 1997, 77, 963– 1032. (17) Willey, R. B. The Morphology of the Stomodeal Nervous System in Periplaneta americana (L.) and Other Blattaria. J. Morphol. 1961, 108, 219-261. (18) Predel, R.; Rapus, J.; Eckert, M. Myoinhibitory Neuropeptides in the American Cockroach. Peptides 2001, 22, 199–208. (19) Clynen, E.; Schoofs, L. Peptidomic Survey of the Locust Neuroendocrine System. Insect Biochem. Mol. Biol. 2009, 39, 491-507. (20) Siegmund, T.; Korge, G. Innervation of the Ring Gland of Drosophila melanogaster. J. Comp. Neurol. 2001, 431, 481–491. (21) Wegener, C.; Reinl, T.; Jänsch, L.; Predel, R. Direct Mass Spectrometric Peptide Profiling and Fragmentation of Larval Peptide Hormone Release Sites in Drosophila melanogaster Reveals TagmaSpecific Peptide Expression and Differential Processing. J. Neurochem. 2006, 96, 1362–1374. (22) Vogt, M. C.; Paeger, L.; Hess, S.; Steculorum, S. M.; Awazawa, M.; Hampel, B.; Neupert, S.; Nicholls, H. T.; Mauer, J.; Hausen, A. C.; et al. Neonatal Insulin Action Impairs Hypothalamic Neurocircuit Formation in Response to Maternal High-Fat Feeding. Cell 2014, 156, 495–509. (23) Trede, D.; Schiffler, S.; Becker, M.; Wirtz, S.; Steinhorst, K.; Strehlow, J.; Aichler, M.; Kobarg, J. H.; Oetjen, J.; Dyatlov, A.; et al. Exploring Three-Dimensional Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry Data: ThreeDimensional Spatial Segmentation of Mouse Kidney. Anal. Chem. 2012, 84 , 6079–6087. (24) Liessem, S.; Ragionieri, L.; Neupert, S.; Büschges, A.; Predel, R. Transcriptomic and Neuropeptidomic Analysis of the Stick Insect, Carausius morosus. J Proteome Res. 2018, 17, 2192-2204. (25) Altelaar, A. F. M.; van Minnen, J.; Jiménez, C. R.; Heeren, R. M. A.; Piersma, S. R. Direct Molecular Imaging of Lymnaea Stagnalis Nervous Tissue at Subcellular Spatial Resolution by Mass Spectrometry. Anal. Chem. 2005, 77, 735–741. (26) Schwartz, S. A.; Reyzer, M. L.; Caprioli, R. M. Direct Tissue Analysis Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry: Practical Aspects of Sample Preparation. J. Mass Spectrom. 2003, 38, 699–708. (27) Seeley, E. H.; Oppenheimer, S. R.; Mi, D.; Chaurand, P.; Caprioli, R. M. Enhancement of Protein Sensitivity for MALDI Imaging Mass Spectrometry after Chemical Treatment of Tissue Sections. J. Am. Soc. Mass Spectrom. 2008, 19, 1069–1077. (28) Meding, S.; Walch, A. MALDI Imaging Mass Spectrometry for Direct Tissue Analysis. Methods Mol. Biol. 2013, 931, 537-546. (29) Pratavieira, M.; Menegasso, A. R. da S.; Esteves, F. G.; Sato, K. U.; Malaspina, O.; Palma, M. S. MALDI Imaging Analysis of Neuropeptides in Africanized Honeybee (Apis mellifera) Brain: Effect of Aggressiveness. J. Proteome Res. 2018, 17, 2358–2369. (30) Cohen, S. L.; Chait, B. T. Influence of Matrix Solution Conditions on the MALDI-MS Analysis of Peptides and Proteins. Anal. Chem. 1996, 68, 31–37. (31) Ong, T.-H.; Romanova, E. V; Roberts-Galbraith, R. H.; Yang, N.;

Page 8 of 9

Zimmerman, T. A.; Collins, J. J.; Lee, J. E.; Kelleher, N. L.; Newmark, P. A.; Sweedler, J. V. Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea Mediterranea. J. Biol. Chem. 2016, 291, 8109–8120. (32) Sadeghi, M.; Vertes, A. Crystallite Size Dependence of Volatilization in Matrix-Assisted Laser Desorption Ionization. Appl. Surf. Sci., 1998, 127-126, 226-234. (33) Dekker, T. J. A.; Jones, E. A.; Corver, W. E.; van Zeijl, R. J. M.; Deelder, A. M.; Tollenaar, R. A. E. M.; Mesker, W. E.; Morreau, H.; McDonnell, L. A. Towards Imaging Metabolic Pathways in Tissues. Anal. Bioanal. Chem. 2015, 407, 2167—2176. (34) Heijs, B.; Holst, S.; Briaire-de Bruijn, I. H.; van Pelt, G. W.; de Ru, A. H.; van Veelen, P. A.; Drake, R. R.; Mehta, A. S.; Mesker, W. E.; Tollenaar, R. A.; Bovée, J.V.M.G.; et al. Multimodal Mass Spectrometry Imaging of N-Glycans and Proteins from the Same Tissue Section. Anal. Chem. 2016, 88, 7745–7753. (35) Gundel, M.; Penzlin, H. Identification of Neuronal Pathways between the Stomatogastric Nervous System and the Retrocerebral Complex of the Cockroach Periplaneta americana (L.). Cell Tissue Res. 1980, 280, 283297. (36) Lococo, D. J.; Tobe, S. S. Neuroanatomy of the Retrocerebral Complex, in Particular the Pars Intercerebralis and Partes Laterales in the Cockroach Diploptera punctata Eschscholtz (Dictyoptera : Blaberidae). Int. J. Insect Morphol. Embryol. 1984, 13, 65-76. (37) Predel, R. Peptidergic Neurohemal System of an Insect: Mass Spectrometric Morphology. J. Comp. Neurol. 2001, 436, 363–375. (38) Nässel, D. R.; Cantera, R.; Karlsson, A. Neurons in the Cockroach Nervous System Reacting with Antisera to the Neuropeptide Leucokinin I. J. Comp. Neurol. 1992, 322, 45–67. (39) Predel, R.; Neupert, S.; Derst, C.; Reinhardt, K.; Wegener, C. Neuropeptidomics of the Bed Bug Cimex lectularius. J. Proteome Res. 2018, 17, 440–454. (40) Schmitt, F.; Vanselow, J. T.; Schlosser, A.; Kahnt, J.; Rössler, W.; Wegener, C. Neuropeptidomics of the Carpenter Ant Camponotus floridanus. J. Proteome Res. 2015, 14, 1504–1514. (41) Verleyen, P.; Baggerman, G.; Wiehart, U.; Schoeters, E.; Van Lommel, A.; De Loof, A.; Schoofs, L. Expression of a Novel Neuropeptide, NVGTLARDFQLPIPNamide, in the Larval and Adult Brain of Drosophila melanogaster. J. Neurochem. 2003, 88, 311–319. (42) Duve, H.; Thorpe, A.; Tobe, S. S. Immunocytochemical Mapping of Neuronal Pathways from Brain to Corpora Cardiaca/Corpora Allata in the Cockroach Diploptera punctata with Antisera against Met-EnkephalinArg6-Gly7-Leu8. Cell Tissue Res. 1991, 263, 285-291. (43) Veenstra, J. A.; Davis, N. T. Localization of Corazonin in the Nervous System of the Cockroach Periplaneta americana. Cell Tissue Res. 1993, 274, 57–64. (44) Predel, R.; Eckert, M.; Pollák, E.; Molnár, L.; Scheibner, O.; Neupert, S. Peptidomics of Identified Neurons Demonstrates a Highly Differentiated Expression Pattern of FXPRLamides in the Neuroendocrine System of an Insect. J. Comp. Neurol. 2007, 500, 498–512. (45) Hankin, J. A.; Barkley, R. M.; Murphy, R. C. Sublimation as a Method of Matrix Application for Mass Spectrometric Imaging. J. Am. Soc. Mass Spectrom. 2007, 18, 1646–1652. (46) Yang, J.; Caprioli, R. M. Matrix Sublimation/Recrystallization for Imaging Proteins by Mass Spectrometry at High Spatial Resolution. Anal. Chem. 2011, 83, 5728–5734. (47) Kompauer, M.; Heiles, S.; Spengler, B. Autofocusing MALDI Mass Spectrometry Imaging of Tissue Sections and 3D Chemical Topography of Nonflat Surfaces. Nat. Methods 2017, 14, 1156-1158.


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Analytical Chemistry For TOC only:


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Enhanced Coverage of Insect Neuropeptides in Tissue Sections by an

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