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Enhanced Coverage of Insect Neuropeptides in Tissue Sections by an Optimized Mass-Spectrometry-Imaging Protocol
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Analytical Chemistry

Cite this: Anal. Chem. 2019, 91, 3, 1980–1988
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https://doi.org/10.1021/acs.analchem.8b04304
Published January 3, 2019

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Abstract

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Mass spectrometry imaging (MSI) of neuropeptides has become a well-established method with the ability to combine spatially resolved information from immunohistochemistry with peptidomics information from mass spectrometric analysis. Several studies have conducted MSI of insect neural tissues; however, these studies did not detect neuropeptide complements in manners comparable to those of conventional peptidomics. The aim of our study was to improve sample preparation so that MSI could provide comprehensive and reproducible neuropeptidomics information. Using the cockroach retrocerebral complex, the presented protocol produces enhanced coverage of neuropeptides at 15 μm spatial resolution, which was 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 neuroscience-related topics, including the study of complex neuropeptide interactions within the CNS.

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Subjects

what are subjects
Neuropeptides are structurally diverse signaling molecules that 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 hormone repositories. The large number of neuropeptides and neuropeptide receptors generally hamper 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 has increasingly been used to analyze the neuropeptidome of the CNS, even to the 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 the honeybee Apis mellifera, small tissue sizes, low peptide abundances, and complex cellular patterns 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 CNSs of insects, but it has limited abilities for visualizing neuropeptides from different precursors in the same sample, even when using fluorochrome-coupled secondary antisera. IHC also usually fails to discriminate between sequence-related precursor products. In insects, this problem is evident 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 the brain peptides of crustaceans. (9,10) Few studies have so far used MALDI-MSI for the detection of insect neuropeptides, and the reported tissue preparations and spatial resolutions (30 μm and greater) are 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 and 10 μm and also reported several mass matches with neuropeptides. (13) In all of these studies, however, mass spectra did not detect neuropeptide contents in a manner comparable to that obtained with the analysis of single dissected neurons or with 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 RCC samples. (15) In comparison with insect brains, whose complexity hampers easy recognition of specific areas by MSI, the organization of the RCC is easier to reconstruct and facilitates reproducibility.
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 are fused posteriorly to a pair of corpora allata. Whereas the corpora allata synthesize sesquiterpenoids (juvenile hormones), the corpora cardiaca exclusively release peptide hormones. Each corpus cardiacum is subdivided antero-dorsally into a glandular part that produces the insect equivalent of glucagon, the adipokinetic hormones (AKHs); (16) the remaining parts of the corpora cardiaca serve as neurohemal release sites 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.

Figure 1

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 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.

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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 distributions of further neuropeptides could be verified for the first time in the RCC.

Experimental Section

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Chemicals and Reagents

α-Cyano-4-hydroxycinnamic 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 (126 mM NaCl, 5.4 mM KCl, 0.17 mM NaH2PO4, 0.22 mM KH2PO4; 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 5 min to ensure dissolution, and then kept at 50 °C to maintain viscosity. For tissue embedding, 400 μL of dissolved gelatin was poured into a handmade aluminum-foil mold with an 8 mm internal diameter and allowed to solidify at room temperature for at least 30 min. Subsequently, the RCCs were placed horizontally 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, 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 s 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). The 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, 20, 30, 40, and 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 the spray-nozzle height (Z-position) was 25 mm.

Immunohistochemistry

Samples were fixed in 4% paraformaldehyde diluted in phosphate-buffered saline (PBS, pH 7.2) at 4 °C for 30 min and subsequently washed three times in PBS for 30 min. The samples were preincubated with 5% normal goat serum dissolved in PBS for 30 min and 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. Following washing (3 × 30 min), the samples were incubated with goat anti-mouse Cy2 (1:500)- and goat anti-rabbit Cy3 (1:3000)-tagged secondary antibodies (Jackson Immuno Research; West Grove, PA) 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 10×/0.45W (NA 0.45) objective using the multitrack mode. Cy2 was excited at 492 nm and emission collected with a BP 505–530 filter, and Cy3 was excited at 543 nm and emission collected via a LP 560 BP filter. Serial optical sections, each 0.9 μm thick, were analyzed 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 and 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 were compared. The extract was measured as a single spectrum using the MSI-acquisition parameters as an imaged area (average of 50 pixels per 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 a 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 a 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 did not obscure the tissue. Ion images were generated using flexImaging v. 5.0 and SCiLS Lab MVS software version 2018a (Bruker Daltonik) with the 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-k-means-with-correlation-distance approach. (23) The default pipeline was used with the following modifications: medium denoising and ±0.30 Da interval width. Ten RCC preparations with the corresponding sections (3–5 sections of each RCC preparation) were analyzed using the MSI protocol.

Quadrupole Orbitrap Mass Spectrometry

The RCC extract was analyzed with a Q-Exactive Plus (Thermo Fisher Scientific; Waltham, MA) 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; Waterloo, Canada) and MaxQuant (v. 1.6.2.10, MPI; Martinsried, Germany), MS2 spectra were compared to an internal database containing known P. americana neuropeptides (14) and the newly annotated neuropeptide-like precursor 1 (NPLP1, MH837510). For both pipelines, the maximum number of PTMs (sulfation of Tyr, C-terminal amidation, cystine formation, oxidation of Met and Trp, pyroglutamyl formation on Glu and Gln, and N-terminal acetylation of 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 the 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 the peptide search using PEAKS, the parent-mass error tolerance was set at 10 ppm, and the fragment-mass error tolerance was set at 0.05 Da. FDR below 1% and fragment spectra with a peptide score (−10 log P) equivalent to a P-value of about 1% were selected and manually reviewed.

Statistics

Paired t tests were used to calculate the effects of different method parameters (GraphPad Prism (v. 5.04); San Diego, CA).

Results and Discussion

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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 an RCC extract detected with the same setup for sample preparation (including the matrix sprayer, matrix composition, and matrix-application procedure) and MALDI-TOF (the same mass analyzer, ionization technique, sample-target device, and instrument settings) that were used for the 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 that 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 an 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 that was much less comprehensive than that obtained from RCC extracts. In fact, only few abundant peptides were detected with weak spatial distributions (data not shown). Therefore, we re-evaluated each experimental step to reach an optimized MSI protocol suitable for 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 a 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 the saline solution and to avoid salt-crystal formation during the embedding–freezing steps. As a general rule, short dissection times of less than 5 min and strict avoidance of direct contact of the 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 toward the periphery.

Step 2: Embedding

The small size of the RCC (approximately 0.5 × 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 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 decreased with lower gelatin concentrations, whereas 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 comparison of peptide distribution in the two parts. We found that 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 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 to −10 °C), and blade cutting angles (5–20°). The best section quality without folding or squeezing tissue sections was achieved with a blade angle of 10°, a temperature of −10 °C, and a tissue thickness of 14–20 μm; although cutting thinner sections was possible, it was 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 tissues and 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 (t test, p = 0.0004, Figure S4). Two consecutive ethanol washes using first 70% (v/v) ethanol/water followed by absolute ethanol for 20 s 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 the detection of intact proteins (28) and slightly modified from those used in the detection of A. mellifera brain neuropeptides. (11,29) For the comparison of peptide coverage, we selected regions of interest (ROIs; 200 × 200 μm) within corpora allata tissue that showed more uniform peptidome in consecutive sections compared with 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 the mass spectra obtained from the washed samples was accompanied by a slight decrease in the resolution of MSI ion maps. For those peptides that were detectable in sections 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 the matrix crystals might have little effect 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 brains (11,29) and flatworms. (31) 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, and number of cycles) were selected on the basis of 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 compared 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 neuropeptide signal intensities in the MSI spectra and a significant increase in 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 Multicopy 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 intensities in the 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 similar distributions of the different FMRF paracopies, which all have specific sequences in P. americana (Figure 2A). The presence of FMRF ion signals was consistent in the mass spectra of the MSI tissue sections (Figure 2B) and the extract samples that had been spotted on ITO glass slides before being analyzed 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 that the distribution of FMRFs in the RCC was resolved, although these peptides showed low signal intensities in the mass spectra from the RCC extracts (Figure 2C).

Figure 2

Figure 2. FMRF paracopies in mass spectra from RCC preparations. (A) MSI from a single tissue section showing the distributions of four FMRFs, suggesting identical spatial distributions of these peptides in the RCC. Section: 20 μm, 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 an RCC extract spotted on an ITO glass slide. The matrix-spraying and MALDI-TOF equipment were exactly the same as those as used for (B). The accuracy of mass matching for peptide assignment was settled at ±0.25 Da.

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Differential Distribution of Neuropeptides in the RCC–SNS

Subsequent to the confirmation that MSI spectra show reliable spatial distributions of neuropeptide paracopies, we analyzed the distributions of all mature neuropeptides detected in our MSI spectra. Altogether, we observed ion signals of 57 mature neuropeptides, a number that matches well 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 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 that described distinct axonal pathways within the seemingly uniform RCC. (35,36) The ion maps only partially matched the few available immunostainings depicting the distribution of neuropeptides in the RCC, but they correspond to the data obtained by direct tissue profiling of parts of the RCC and SNS. (37) A brief summary of the distribution patterns of neuropeptides in the RCC–SNS, as revealed by MSI, is given hereafter.

Figure 3

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 an overview of the architecture of RCC–SNS). Scale bar (white): 600 μm, section thicknesses: (A–I) 20 μm and (J–L) 14 μm.

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Myosuppressin and sk genes are both expressed in neurosecretory cells of the pars intercerebralis in the protocerebrum with projection into the RCC via NCC-1. The neuropeptides from these genes showed distinct accumulations within the RCC (Figure 3A,B) that were 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, whereas MIPs were more abundant along the SNS (Figure 3D,E). As 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 part of the corpora cardiaca only (Figure 3L).
We also observed the spatial distributions of neuropeptides not previously described by mass spectrometry in the RCC of P. americana, such as allatotropin (AT; Figures 3I and 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 on the basis of the MSI information, it seems possible that AT reaches the corpora cardiaca through 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 distributions mostly restricted to the neurohemal part of corpora cardiaca (Figures 3H and S1 and Table S1).
Corazonin and AstA peptides, both of which were detected using IHC in cells of the pars lateralis of the protocerebrum with projection via 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 the 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 the MSI images from consecutive sections (Figure 4). The resulting data confirmed that AstA and corazonin indeed have different spatial distributions along the RCC (see also Figure S6).

Figure 4

Figure 4. Distribution of corazonin and AstA analyzed in serial RCC sections by (A) immunohistochemistry and (B) MSI (the more peripheral section). Data obtained by both methods confirmed the different spatial distributions 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%. The accuracy of mass matching for peptide assignment was settled at ±0.25 Da.

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Differential Prohormone Processing

An advantage of MSI is the capability of detecting differential prohormone processing. In P. americana, differential processing is only demonstrated for the PK precursor. Although the full set of PKs is processed in cell clusters of the SEG with projections to the RCC via NCA-2, few cells in the brain with projections into the RCC via NCC-1 do not process PK-1, and mass spectra of 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, whereas all PKs were prominent in the neurohemal part of the RCC near the entrance of NCA-2 and in the corpora allata.

Figure 5

Figure 5. (A) Ion maps of four PKs indicating differential processing of the PK precursor. (B) Four PKs detected in the posterior part of the RCC, which mostly contains PKs processed in cells of the SEG. (C) Anterior corpus cardiacum tissue, which receives neuropeptides from the brain, showing no 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%). The accuracies of mass matching for peptide assignment were settled at ±0.25 Da for PK-2, -3, and -4 and at ±0.001 Da for PK-1. Tissue sections were not washed with ethanol prior to matrix spraying.

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Two of the PKs (PK-1 and PK-3) have ion signals with similar masses to those of 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 showed 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 the main compartments within the RCC–SNS (Figure 6). These regions correspond to the corpora allata and adjoining nervi corporis allati-1, the corpora cardiaca glandular area, the corpora cardiaca neurohemal area, and the 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 the SNS.

Figure 6

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) and the glandular and neurohemal corpora cardiaca (CC). The neurohemal part of the corpora cardiaca is further subdivided into three subcompartments.

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Conclusions

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This study provides an MSI workflow for analysis of neuropeptides in insect neuroendocrine tissues that 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 that 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 or spatial resolutions (e.g., MALDI-FT-ICR and AP-SMALDI-Orbitrap). Enhanced lateral resolution may be possible particularly in combination with spraying devices that result in smaller matrix-crystal sizes 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 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.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b04304.

  • List of mature neuropeptides from 15 precursor genes of P. americana; quadrupole orbitrap MS2 spectra of P. americana neuropeptides; workflow for MSI sample preparation optimized for insect neuroendocrine tissue (RCC); comparison of peptide coverage in tissue sections dried for 1 or 12 h before washing; comparison of peptide coverage in tissue sections with and without successive ethanol washes; comparison of peptide coverage in tissue sections after matrix spraying (5 mg/mL CHCA in 50% ACN/H2O) with matrix solution containing 0.1 or 2% TFA; distribution of allatostatinA-11, corazonin, and allatotropin in consecutive RCC sections; MALDI-TOF direct tissue profiling of a dissected nervus corporis cardiaci 1; and discrimination between mass-similar neuropeptides (PDF)

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Author Information

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  • Corresponding Authors
  • Authors
    • Sander Liessem - Department for Biology, Institute of Zoology, University of Cologne, 50674 Cologne, Germany;  Orcidhttp://orcid.org/0000-0002-7073-2659
    • Michael Becker - Bruker Daltonik GmbH, Fahrenheitstraße 4, 28359 Bremen, Germany;  Present Address: M.B.: Boehringer Ingelheim Pharma GmbH & Company KG, 88397 Biberach an der Riss, Germany
    • Sören-Oliver Deininger - Bruker Daltonik GmbH, Fahrenheitstraße 4, 28359 Bremen, Germany
  • Author Contributions

    A.L. and L.R. contributed equally. All authors contributed to the writing and have given approval to the final version of the manuscript.

  • Notes
    The authors declare the following competing financial interest(s): A.L. and S.O.D. were employees of Bruker Daltonik GmbH for the duration of this study. M.B. was an employee of Bruker for part of this study.

Acknowledgments

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This project was supported by a European Commission Horizon2020 Research and Innovation Grant 634361 (nEUROSTRESSPEP); the German Research Foundation (PR 766/11-1); and the 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

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This article references 47 other publications.

  1. 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,  DOI: 10.1016/j.bbrc.2005.07.038
    Google Scholar
    1
    The Drosophila gene CG9918 codes for a pyrokinin-1 receptor
    Cazzamali, Giuseppe; Torp, Malene; Hauser, Frank; Williamson, Michael; Grimmelikhuijzen, Cornelis J. P.
    Biochemical and Biophysical Research Communications (2005), 335 (1), 14-19CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)
    The database from the Drosophila Genome Project contains a gene, CG9918, annotated to code for a G protein-coupled receptor. We cloned the cDNA of this gene and functionally expressed it in Chinese hamster ovary cells. We tested a library of about 25 Drosophila and other insect neuropeptides, and seven insect biogenic amines on the expressed receptor and found that it was activated by low concns. of the Drosophila neuropeptide, pyrokinin-1 (TGPSASSGLWFGPRLamide; EC50, 5 × 10-8 M). The receptor was also activated by other Drosophila neuropeptides, terminating with the sequence PRLamide (Hug-γ, ecdysis-triggering-hormone-1, pyrokinin-2), but in these cases about six to eight times higher concns. were needed. The receptor was not activated by Drosophila neuropeptides, contg. a C-terminal PRIamide sequence (such as ecdysis-triggering-hormone-2), or PRVamide (such as capa-1 and -2), or other neuropeptides and biogenic amines not related to the pyrokinins. This paper is the first conclusive report that CG9918 is a Drosophila pyrokinin-1 receptor gene.
  2. 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,  DOI: 10.1016/S0006-291X(02)02709-2
    Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Smith, M. E. POMC Opioid Peptides. In Handbook of Biologically Active Peptides; Kastin, A. J., Ed.; Elsevier: San Diego, 2006; pp 1325– 1331.
    Google Scholar
    There is no corresponding record for this reference.
  4. 4
    Rubakhin, S. S.; Sweedler, J. V. Characterizing Peptides in Individual Mammalian Cells Using Mass Spectrometry. Nat. Protoc. 2007, 2, 1987– 97,  DOI: 10.1038/nprot.2007.277
    Google Scholar
    4
    Characterizing peptides in individual mammalian cells using mass spectrometry
    Rubakhin, Stanislav S.; Sweedler, Jonathan V.
    Nature Protocols (2007), 2 (8), 1987-1997CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
    Cell-to-cell chem. signaling plays multiple roles in coordinating the activity of the functional elements of an organism, with these elements ranging from a three-neuron reflex circuit to the entire animal. In recent years, single-cell mass spectrometry (MS) has enabled the discovery of cell-to-cell signaling mols. from the nervous system of a no. of invertebrates. The authors describe a protocol for analyzing individual cells from rat pituitary using matrix-assisted laser desorption/ionization MS. Each step in the sample prepn. process, including cell stabilization, isolation, sample prepn., signal acquisition and data interpretation, is detailed here. Although the authors employ this method to investigate peptides in individual pituitary cells, it can be adapted to other cell types and even subcellular sections from a range of animals. This protocol allows one to obtain 20-30 individual cell samples and acquire mass spectra from them in a single day.
  5. 5
    Neupert, S.; Johard, H. A. D.; Nässel, D. R.; Predel, R. Single-Cell Peptidomics of Drosophila melanogaster Neurons Identified by Gal4-Driven Fluorescence. Anal. Chem. 2007, 79, 3690– 3694,  DOI: 10.1021/ac062411p
    Google Scholar
    5
    Single-Cell Peptidomics of Drosophila melanogaster Neurons Identified by Gal4-Driven Fluorescence
    Neupert, Susanne; Johard, Helena A. D.; Naessel, Dick R.; Predel, Reinhard
    Analytical Chemistry (Washington, DC, United States) (2007), 79 (10), 3690-3694CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Neuropeptides are widespread signal mols. that display a great chem. and functional diversity. Predictions of neuropeptide cleavage from precursor proteins are not always correct, and thus, biochem. identification is essential. Single-cell anal. is valuable to identify peptides processed from a single precursor, but also to det. coexpression of further neuropeptides from other precursors. The authors have developed an approach to isolate single identified neurons from the fruit fly Drosophila melanogaster for mass spectrometric anal. By using Gal4 promoter lines to drive green fluorescent protein under UAS control, the authors identified specific peptidergic neurons. These neurons were isolated singly under a fluorescence microscope and subjected to MALDI-TOF mass spectrometry. Two Gal4 lines were used here to identify pigment-dispersing factor (PDF) and hugin-expressing neurons. The authors found that the large PDF expressing clock neurons only give rise to a single peptide, PDF. The three different classes of hugin expressing neurons all display the same mass signal, identical to pyrokinin-2. The other peptide predicted from the hugin precursor, hugin γ, was not detected in any of the cells. Single-cell peptidomics is a powerful tool in Drosophila neuroscience since Gal4 drivers can be produced for all known neuropeptide genes and thus provide detailed information about neuropeptide complements in neurons of interest.
  6. 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,  DOI: 10.1007/s00216-010-3723-7
    Google Scholar
    6
    Mass spectral imaging and profiling of neuropeptides at the organ and cellular domains
    Chen, Ruibing; Li, Lingjun
    Analytical and Bioanalytical Chemistry (2010), 397 (8), 3185-3193CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    A review. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a rapid and sensitive anal. method that is well suited for detg. mol. wts. of peptides and proteins from complex samples. MALDI-MS can be used to profile the peptides and proteins from single-cell and small tissue samples without the need for extensive sample prepn. Furthermore, the recently developed MALDI imaging technique enables mapping of the spatial distribution of signaling mols. in tissue samples. Several examples of signaling mol. anal. at the single-cell and single-organ levels using MALDI-MS technol. are highlighted followed by an outlook of future directions.
  7. 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,  DOI: 10.1021/acs.analchem.7b04733
    Google Scholar
    7
    Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights
    Buchberger, Amanda Rae; DeLaney, Kellen; Johnson, Jillian; Li, Lingjun
    Analytical Chemistry (Washington, DC, United States) (2018), 90 (1), 240-265CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A review. Mass spectrometry imaging (MSI) is a powerful tool that enables untargeted studies into the spatial distribution of mol. species in a variety of samples. It has the capability to image thousands of mols., such as metabolites, lipids, peptides, proteins, and glycans, in a single expt. without labeling. The combination of information gained from mass spectrometry (MS) and visualization of spatial distributions in thin sample sections makes this a valuable chem. anal. tool useful for biol. specimen characterization. After minimal but careful sample prepn., the general setup of an MSI expt. involves defining an (x, y) grid over the surface of the sample, with the grid area chosen by the user. The mass spectrometer then ionizes the mols. on the surface of the sample and collects a mass spectrum at each pixel on the section, with the resulting spatial resoln. defined by the pixel size. After collecting the spectra, computational software can be used to select an individual mass-to-charge (m/z) value, and the intensity of the m/z is extd. from each pixel's spectrum. These intensities are then combined into a heat map image depicting the relative distribution of that m/z value throughout the sample's surface. In order to det. the identity of a specific m/z value, tandem MS (MS/MS) fragmentation can be performed on ions from each pixel, and the fragments can be used to piece together the structure of the unknown mol. Otherwise, the mol. can be identified based on its intact mass by accurate mass matching to databases of known mols. within a certain mass error range. Overall, the aim of this review is to provide an informative resource for those in the MSI community who are interested in improving MSI data quality and anal. or using MSI for novel applications. Particularly, advances from the last two years in sample prepn., instrumentation, quantitation, statistics, and multi-modal imaging that have allowed MSI to emerge as a powerful technique in various biomedical applications including clin. settings are discussed. Also, several novel biol. applications are highlighted to demonstrate the potential for the future of the MSI field.
  8. 8
    Spengler, B. Mass Spectrometry Imaging of Biomolecular Information. Anal. Chem. 2015, 87, 64– 82,  DOI: 10.1021/ac504543v
    Google Scholar
    8
    Mass Spectrometry Imaging of Biomolecular Information
    Spengler, Bernhard
    Analytical Chemistry (Washington, DC, United States) (2015), 87 (1), 64-82CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A review. Mass spectrometry imaging (MSI) has gone through a significant change over the last three years, the time span of this review. The predominant mass spectrometric method in biomol. imaging has been matrix-assisted laser desorption/ionization (MALDI). MALDI MSI, after its first introduction in 1994 [1] has suffered from a phase of rather unsystematic use, without validation on an anal.-science level. Not meeting a high std. of validity for these colorful pictures, MSI reached a dangerous state of being suspected as a non-trustful method, useless for precise biomedical or mol.-biol. research. With the introduction of new, highly sensitive, highly accurate methods of data acquisition and imaging, which were based on high mass resoln., high mass accuracy and high lateral resoln., MALDI MSI recovered and developed as a powerful, versatile and valid method in bioanal. sciences [2-4]. At the same time alternative ionization methods came into the game, providing imaging capability with reduced sample prepn. requirements and specific anal. properties. The last three years have thus been characterized by developments and applications in biomol. imaging, which are unprecedented not only in the view of mass spectrometrists.
  9. 9
    Chen, R.; Cape, S. S.; Sturm, R. M.; Li, L. Mass Spectrometric Imaging of Neuropeptides in Decapod Crustacean Neuronal Tissues. Methods Mol. Biol. 2010, 656, 451– 463,  DOI: 10.1007/978-1-60761-746-4_26
    Google Scholar
    9
    Mass spectrometric imaging of neuropeptides in decapod crustacean neuronal tissues
    Chen, Ruibing; Cape, Stephanie S.; Sturm, Robert M.; Li, Lingjun
    Methods in Molecular Biology (New York, NY, United States) (2010), 656 (Mass Spectrometry Imaging), 451-463CODEN: MMBIED; ISSN:1064-3745. (Springer)
    The emerging technol. mass spectrometric imaging (MSI) provides an attractive opportunity to detect and probe the mol. content of tissues in an anatomical context. This powerful methodol. has been applied extensively to the localization of proteins, peptides, pharmaceuticals, metabolites, lipids, and other biol. and chem. compds. in tissues. Herein, we present a method developed specifically for mapping neuropeptides in crustacean neuronal tissues. Both cryostat tissue sectioning and whole-mount tissue blotting techniques are highlighted. Careful sample prepn. is essential for obtaining sufficient analyte/matrix mixing while retaining the spatial localization of the neuropeptides. Several matrix application app. and techniques are described and compared. Furthermore, three-dimensional (3D) imaging has been developed to provide detailed information about the distribution of neuropeptides within 3D structure of a crustacean brain.
  10. 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,  DOI: 10.1021/pr900736t
    Google Scholar
    10
    Mass Spectral Analysis of Neuropeptide Expression and Distribution in the Nervous System of the Lobster Homarus americanus
    Chen, Ruibing; Jiang, Xiaoyue; Prieto Conaway, Maria C.; Mohtashemi, Iman; Hui, Limei; Viner, Rosa; Li, Lingjun
    Journal of Proteome Research (2010), 9 (2), 818-832CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    The lobster Homarus americanus has long served as an important animal model for electrophysiol. and behavioral studies. Using this model, we performed a comprehensive investigation of the neuropeptide expression and their localization in the nervous system, which provides useful insights for further understanding of their biol. functions. Using nanoLC ESI Q-TOF MS/MS and 3 types of MALDI instruments, we analyzed the neuropeptide complements in a major neuroendocrine structure, pericardial organ. A total of 57 putative neuropeptides were identified and 18 of them were de novo sequenced. Using direct tissue/ext. anal. and bioinformatics software SpecPlot, we charted the global distribution of neuropeptides throughout the nervous system in H. americanus. Furthermore, we also mapped the localization of several neuropeptide families in the brain by high mass resoln. and high mass accuracy mass spectrometric imaging (MSI) using a MALDI LTQ Orbitrap mass spectrometer. We have also compared the utility and instrument performance of multiple mass spectrometers for neuropeptide anal. in terms of peptidome coverage, sensitivity, mass spectral resoln. and capability for de novo sequencing.
  11. 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,  DOI: 10.1021/pr500224b
    Google Scholar
    11
    MALDI Imaging Analysis of Neuropeptides in the Africanized Honeybee (Apis mellifera) Brain: Effect of Ontogeny
    Pratavieira, Marcel; da Silva Menegasso, Anally Ribeiro; Garcia, Ana Maria Caviquioli; dos Santos, Diego Simoes; Gomes, Paulo Cesar; Malaspina, Osmar; Palma, Mario Sergio
    Journal of Proteome Research (2014), 13 (6), 3054-3064CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    The occurrence and spatial distribution of the neuropeptides AmTRP-5 and AST-1 in the honeybee brain were monitored via MALDI spectral imaging according to the ontogeny of Africanized Apis mellifera. The levels of these peptides increased in the brains of 0-15 day old honeybees, and this increase was accompanied by an increase in the no. of in-hive activities performed by the nurse bees, followed by a decrease in the period from 15 to 25 days of age, in which the workers began to perform activities outside the nest (guarding and foraging). The results obtained in the present investigation suggest that AmTRP-5 acts in the upper region of both pedunculi of young workers, possibly regulating the cell cleaning and brood capping activities. Meanwhile, the localized occurrence of AmTRP-5 and AST-1 in the antennal lobes, subesophageal ganglion, upper region of the medulla, both lobula, and α- and β-lobes of both brain hemispheres in 20-25 day old workers suggest that the action of both neuropeptides in these regions may be related to their localized actions in these regions, regulating foraging and guarding activities. Thus, these neuropeptides appear to have some functions in the honeybee brain that are specifically related to the age-related division of labor.
  12. 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,  DOI: 10.1007/978-1-60761-746-4_25
    Google Scholar
    12
    Imaging of similar mass neuropeptides in neuronal tissue by enhanced resolution MALDI MS with an ion trap - Orbitrap hybrid instrument
    Verhaert, Peter D. E. M.; Pinkse, Martijn W. H.; Strupat, Kerstin; Conaway, Maria C. Prieto
    Methods in Molecular Biology (New York, NY, United States) (2010), 656 (Mass Spectrometry Imaging), 433-449CODEN: MMBIED; ISSN:1064-3745. (Springer)
    Several mass spectrometry imaging (MSI) procedures are used to localize physiol. active peptides in neuronal tissue from American cockroach (Periplaneta americana) neurosecretory organs. We report how to use this model system to assess, for the first time, the performance of the MALDI LTQ Orbitrap XL mass spectrometer to perform MSI of secretory neuropeptides. The method involves the following steps: (1) rapid dissecting of neurosecretory tissue (i.e., insect neurohemal organ) in isotonic sucrose soln.; (2) mounting the tissue on a glass slide; (3) controlled spraying of the air-dried tissue with concd. MALDI matrix soln.; (4) loading specimen into the MALDI source of a MSn system equipped with an Orbitrap analyzer; (5) setting-up MSI methods by detg. tissue areas of interest, spatial resoln., mol. mass range, and mol. mass resoln.; (6) acquiring mass spectra; (7) analyzing data using ImageQuest MSI software to generate (single or composite) images of the distribution of peptide(s) of interest; (8) confirming the identity of selected peptides by MS2 and/or MSn sequencing directly from imaged tissue sample. The results illustrate that high mass accuracy and high mass resolving power of the Orbitrap analyzer are achievable in analyses directly from tissue, such as in MSI expts. Moreover the mass spectrometric instrumentation evaluated allows for both peptide localization and peptide identification/sequencing directly from tissue.
  13. 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,  DOI: 10.1016/j.ijms.2017.04.001
    Google Scholar
    There is no corresponding record for this reference.
  14. 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. 2012, 520, 694– 716,  DOI: 10.1002/cne.22745
    Google Scholar
    14
    Toward a single-cell-based analysis of neuropeptide expression in Periplaneta americana antennal lobe neurons
    Neupert, Susanne; Fusca, Debora; Schachtner, Joachim; Kloppenburg, Peter; Predel, Reinhard
    Journal of Comparative Neurology (2012), 520 (4), 694-716CODEN: JCNEAM; ISSN:0021-9967. (Wiley-Blackwell)
    A multitude of potential neurotransmitters and neuromodulators, including peptides, have been detected in the antennal lobe (AL), the first synaptic relay of the central olfactory pathway in the insect brain. However, the functional role of neuropeptides in this system has yet to be revealed. An important prerequisite to understanding the role of neuropeptides is to match the functionally different cell types in the AL with their peptide profiles by using electrophysiol. recordings combined with immunocytochem. studies and/or single-cell mass spectrometry. The olfactory system of Periplaneta americana is particularly well suited to accomplish this goal because several physiol. distinct neuron types can be unequivocally identified. With the aim to analyze the neuropeptide inventory of the P. americana AL, this study is an essential step in this direction. First, the authors systematically analyzed different parts of the AL by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry to obtain the complete set of neuropeptides present. Altogether, 56 ion signals could be assigned to products of 10 neuropeptide genes (allatostatins A, B, C, SIFamide, allatotropin, FMRFamide-related peptides [myosuppressin, short neuropeptides F, extended FMRFamides], crustacean cardioactive peptide, tachykinin-related peptides). In a second step, a combination of immunocytochem. and mass spectrometric profiling of defined AL compartments was used to reveal the spatial distribution of neuropeptide-contg. cells. Finally, the authors demonstrated the feasibility of MALDI-TOF mass spectrometric profiling of single AL neurons, which is an important precondition for combining electrophysiol. with peptide profiling at the single-cell level. J. Comp. Neurol., 2012. © 2011 Wiley Periodicals, Inc.
  15. 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-Year-Old Formalin-Fixed, Paraffin-Embedded Tissue by Mass Spectrometry Imaging. Anal. Chem. 2018, 90, 9272– 9280,  DOI: 10.1021/acs.analchem.8b01838
    Google Scholar
    15
    Digestion-Free Analysis of Peptides from 30-year-old Formalin-Fixed, Paraffin-Embedded Tissue by Mass Spectrometry Imaging
    Paine, Martin R. L.; Ellis, Shane R.; Maloney, Dan; Heeren, Ron M. A.; Verhaert, Peter D. E. M.
    Analytical Chemistry (Washington, DC, United States) (2018), 90 (15), 9272-9280CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Formalin-fixed neuroendocrine tissues from American cockroaches (Periplaneta americana) embedded in paraffin more than 30 years ago were recently analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), to reveal the histol. localization of more than 20 peptide ions. These represented protonated, and other cationic species of, at least, 14 known neuropeptides. The characterization of peptides in such historical samples was made possible by a novel sample prepn. protocol rendering the endogenous peptides readily amenable to MSI anal. The protocol comprises brief deparaffinization steps involving xylene and ethanol, and is further devoid of conventional aq. washing, buffer incubations, or antigen retrieval steps. Endogenous secretory peptides that are typically highly sol. are therefore retained in-tissue with this protocol. The method is fully "top-down", i.e., without laborious in situ enzymic digestion that typically disturbs the detection of low-abundance endogenous peptides by MSI. Peptide identifications were supported by accurate mass, on-tissue tandem MS analyses, and by earlier MALDI-MSI results reported for freshly prepd. P. americana samples. In contrast to earlier literature accounts stating that MALDI-MSI detection of endogenous peptides is possible only in fresh or freshly frozen tissues, or exceptionally, in formalin-fixed, paraffin-embedded (FFPE) material of less than 1 yr old, the authors demonstrate that MALDI-MSI works for endogenous peptides in FFPE tissue of up to 30 years old. The findings put forward a useful method for digestion-free, high-throughput anal. of endogenous peptides from FFPE samples and offer the potential for reinvestigating archived and historically interesting FFPE material, such as those stored in hospital biobanks.
  16. 16
    Gäde, G.; Hoffmann, K. H.; Spring, J. H. Hormonal Regulation in Insects: Facts, Gaps, and Future Directions. Physiol. Rev. 1997, 77, 963– 1032,  DOI: 10.1152/physrev.1997.77.4.963
    Google Scholar
    There is no corresponding record for this reference.
  17. 17
    Willey, R. B. The Morphology of the Stomodeal Nervous System in Periplaneta americana (L.) and Other Blattaria. J. Morphol. 1961, 108, 219– 261,  DOI: 10.1002/jmor.1051080207
    Google Scholar
    17
    The morphology of the stomodeal nervous system in Periplaneta americana (L.) and other blattaria
    WILLEY R B
    Journal of morphology (1961), 108 (), 219-61 ISSN:0362-2525.
    There is no expanded citation for this reference.
  18. 18
    Predel, R.; Rapus, J.; Eckert, M. Myoinhibitory Neuropeptides in the American Cockroach. Peptides 2001, 22, 199– 208,  DOI: 10.1016/S0196-9781(00)00383-1
    Google Scholar
    18
    Myoinhibitory neuropeptides in the American cockroach
    Predel, R.; Rapus, J.; Eckert, M.
    Peptides (New York) (2001), 22 (2), 199-208CODEN: PPTDD5; ISSN:0196-9781. (Elsevier Science Inc.)
    A large no. of myostimulatory neuropeptides from neurohemal organs of the American cockroach (Periplaneta americana) have been described since 1989. These peptides, isolated from the retrocerebral complex and abdominal perisympathetic organs, are thought to be released as hormones. To study the coordinated action of these neuropeptides in the regulation of visceral muscle activity, it might be necessary to include myoinhibitors as well, however, not a single myoinhibitory neuropeptide of the American cockroach has been described so far. To fill this gap, the authors describe the isolation of LMS (leucomyosuppressin) and Pea-MIP (myoinhibitory peptide) from neurohemal organs of the American cockroach. LMS was very effective in inhibiting phasic activity of all visceral muscles tested. It was found in the corpora cardiaca of different species of cockroaches, as well as in related insect groups, including mantids and termites. Pea-MIP which is strongly accumulated in the corpora cardiaca was not detected with a muscle bioassay system but when searching for tryptophane-contg. peptides using a diode-array detector. This peptide caused only a moderate inhibition in visceral muscle assays. The distribution of Pea-MIP in neurohemal organs and cells supplying these organs with Pea-MIP immunoreactive material, is described. Addnl. to LMS and Pea-MIP, a member of the allatostatin peptide family, known to exhibit inhibitory properties in other insects, was tested in visceral muscle assays. This allatostatin was highly effective in inhibiting spontaneous activity of the foregut, but not of other tested visceral muscles of the American cockroach.
  19. 19
    Clynen, E.; Schoofs, L. Peptidomic Survey of the Locust Neuroendocrine System. Insect Biochem. Mol. Biol. 2009, 39, 491– 507,  DOI: 10.1016/j.ibmb.2009.06.001
    Google Scholar
    19
    Peptidomic survey of the locust neuroendocrine system
    Clynen, Elke; Schoofs, Liliane
    Insect Biochemistry and Molecular Biology (2009), 39 (8), 491-507CODEN: IBMBES; ISSN:0965-1748. (Elsevier Ltd.)
    Neuropeptides are important controlling agents in animal physiol. To understand their role and the ways in which neuropeptides behave and interact with one another, information on their time and sites of expression is required. The authors here used a combination of MALDI-TOF and ESI-Q-TOF mass spectrometry to make an inventory of the peptidome of different parts (ganglia and nerves) of the central nervous system from the desert locust Schistocerca gregaria and the African migratory locust Locusta migratoria. This way, the authors analyzed the brain, subesophageal ganglion, retrocerebral complex, stomatogastric nervous system, thoracic ganglia, abdominal ganglia and abdominal neurohemal organs. The result is an overview of the distribution of sixteen neuropeptide families, i.e., pyrokinins, pyrokinin-like peptides, periviscerokinins, tachykinins, allatotropin, accessory gland myotropin, FLRFamide, (short) neuropeptide F, allatostatins, insulin-related peptide co-peptide, ion-transport peptide co-peptide, corazonin, sulfakinin, orcokinin, hypertrehalosemic hormone and adipokinetic hormones (joining peptides) throughout the locust neuroendocrine system.
  20. 20
    Siegmund, T.; Korge, G. Innervation of the Ring Gland of Drosophila melanogaster. J. Comp. Neurol. 2001, 431, 481– 491,  DOI: 10.1002/1096-9861(20010319)431:4<481::AID-CNE1084>3.0.CO;2-7
    Google Scholar
    20
    Innervation of the ring gland of Drosophila melanogaster
    Siegmund T; Korge G
    The Journal of comparative neurology (2001), 431 (4), 481-91 ISSN:0021-9967.
    In insects, peptidergic neurons of the central nervous system regulate the synthesis of the main developmental hormones. Neuropeptides involved in this neuroendocrine cascade have been identified in lepidopterans and dictyopterans. Since these organisms are not suitable for genetic research, we identified peptidergic brain neurons innervating the ring gland in Drosophila melanogaster. In larvae of Drosophila, ecdysteroids and juvenile hormones are produced by the ring gland, which is composed of the prothoracic gland, the corpus allatum, and the corpora cardiaca. Using the GAL4 enhancer trap system, we mapped those neurons of the central nervous system that innervate the ring gland. Eleven groups of neurosecretory neurons and their target tissues were identified. Five neurons of the lateral protocerebrum directly innervate the prothoracic gland or corpus allatum cells of the ring gland and are believed to regulate ecdysteroid and juvenile hormone titers. Axons of the circadian pacemaker neurons project onto dendritic fields of these five neurons. This connection might be the neuronal substrate of the circadian rhythms of molting and metamorphosis in Drosophila. Most of the neurons presented here have not been described before. The enhancer trap lines labeling them will be valuable tools for the analysis of neuronal as well as genetic regulation in insect development.
  21. 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 Tagma-Specific Peptide Expression and Differential Processing. J. Neurochem. 2006, 96, 1362– 1374,  DOI: 10.1111/j.1471-4159.2005.03634.x
    Google Scholar
    21
    Direct mass spectrometric peptide profiling and fragmentation of larval peptide hormone release sites in Drosophila melanogaster reveals tagma-specific peptide expression and differential processing
    Wegener, Christian; Reinl, Tobias; Jaensch, Lothar; Predel, Reinhard
    Journal of Neurochemistry (2006), 96 (5), 1362-1374CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)
    Regulatory peptides represent a diverse group of messenger mols. In insects, they are produced by endocrine cells as well as secretory neurons within the CNS. Many regulatory peptides are released as hormones into the hemolymph to regulate, for example, diuresis, heartbeat or ecdysis behavior. Hormonal release of neuropeptides takes place at specialized organs, so-called neurohemal organs. The authors have performed a mass spectrometric characterization of the peptide complement of the main neurohemal organs and endocrine cells of the Drosophila melanogaster larva to gain insight into the hormonal communication possibilities of the fruit fly. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and MALDI-TOF-TOF tandem mass spectrometry, the authors detected 23 different peptides of which five were unpredicted by previous genome screenings. The authors also found a hitherto unknown peptide product of the capa gene in the ring gland and transverse nerves, suggesting that it might be released as hormone. The authors' results show that the peptidome of the neurohemal organs is tagma-specific and does not change during metamorphosis. The authors also provide evidence for the first case of differential prohormone processing in Drosophila.
  22. 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. Neonatal Insulin Action Impairs Hypothalamic Neurocircuit Formation in Response to Maternal High-Fat Feeding. Cell 2014, 156, 495– 509,  DOI: 10.1016/j.cell.2014.01.008
    Google Scholar
    22
    Neonatal Insulin Action Impairs Hypothalamic Neurocircuit Formation in Response to Maternal High-Fat Feeding
    Vogt, Merly C.; Paeger, Lars; Hess, Simon; Steculorum, Sophie M.; Awazawa, Motoharu; Hampel, Brigitte; Neupert, Susanne; Nicholls, Hayley T.; Mauer, Jan; Hausen, A. Christine; Predel, Reinhard; Kloppenburg, Peter; Horvath, Tamas L.; Bruening, Jens C.
    Cell (Cambridge, MA, United States) (2014), 156 (3), 495-509CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Maternal metabolic homeostasis exerts long-term effects on the offspring's health outcomes. Maternal high-fat diet (HFD) feeding during lactation predisposes the offspring for obesity and impaired glucose homeostasis in mice, which is assocd. with an impairment of the hypothalamic melanocortin circuitry. Whereas the no. and neuropeptide expression of anorexigenic proopiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP) neurons, electrophysiol. properties of POMC neurons, and posttranslational processing of POMC remain unaffected in response to maternal HFD feeding during lactation, the formation of POMC and AgRP projections to hypothalamic target sites is severely impaired. Abrogating insulin action in POMC neurons of the offspring prevents altered POMC projections to the preautonomic paraventricular nucleus of the hypothalamus (PVH), pancreatic parasympathetic innervation, and impaired glucose-stimulated insulin secretion in response to maternal overnutrition. These expts. reveal a crit. timing, when altered maternal metab. disrupts metabolic homeostasis in the offspring via impairing neuronal projections, and show that abnormal insulin signaling contributes to this effect.
  23. 23
    Trede, D.; Schiffler, S.; Becker, M.; Wirtz, S.; Steinhorst, K.; Strehlow, J.; Aichler, M.; Kobarg, J. H.; Oetjen, J.; Dyatlov, A. Exploring Three-Dimensional Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry Data: Three-Dimensional Spatial Segmentation of Mouse Kidney. Anal. Chem. 2012, 84, 6079– 6087,  DOI: 10.1021/ac300673y
    Google Scholar
    23
    Exploring Three-Dimensional Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry Data: Three-Dimensional Spatial Segmentation of Mouse Kidney
    Trede, Dennis; Schiffler, Stefan; Becker, Michael; Wirtz, Stefan; Steinhorst, Klaus; Strehlow, Jan; Aichler, Michaela; Kobarg, Jan Hendrik; Oetjen, Janina; Dyatlov, Andrey; Heldmann, Stefan; Walch, Axel; Thiele, Herbert; Maass, Peter; Alexandrov, Theodore
    Analytical Chemistry (Washington, DC, United States) (2012), 84 (14), 6079-6087CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Three-dimensional (3D) imaging has a significant impact on many challenges of life sciences. Three-dimensional matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is an emerging label-free bioanal. technique capturing the spatial distribution of hundreds of mol. compds. in 3D by providing a MALDI mass spectrum for each spatial point of a 3D sample. Currently, 3D MALDI-IMS cannot tap its full potential due to the lack efficient computational methods for constructing, processing, and visualizing large and complex 3D MALDI-IMS data. We present a new pipeline of efficient computational methods, which enables anal. and interpretation of a 3D MALDI-IMS data set. Construction of a MALDI-IMS data set was done according to the state-of-the-art protocols and involved sample prepn., spectra acquisition, spectra preprocessing, and registration of serial sections. For anal. and interpretation of 3D MALDI-IMS data, we applied the spatial segmentation approach which is well-accepted in anal. of two-dimensional (2D) MALDI-IMS data. In line with 2D data anal., we used edge-preserving 3D image denoising prior to segmentation to reduce strong and chaotic spectrum-to-spectrum variation. For segmentation, we used an efficient clustering method, called bisecting k-means, which is optimized for hierarchical clustering of a large 3D MALDI-IMS data set. Using the proposed pipeline, we analyzed a central part of a mouse kidney using 33 serial sections of 3.5 μm thickness after the PAXgene tissue fixation and paraffin embedding. For each serial section, a 2D MALDI-IMS data set was acquired following the std. protocols with the high spatial resoln. of 50 μm. Altogether, 512 495 mass spectra were acquired that corresponds to approx. 50 gigabytes of data. After registration of serial sections into a 3D data set, our computational pipeline allowed us to reveal the 3D kidney anatomical structure based on mass spectrometry data only. Finally, automated anal. discovered mol. masses colocalized with major anatomical regions. In the same way, the proposed pipeline can be used for anal. and interpretation of any 3D MALDI-IMS data set in particular of pathol. cases.
  24. 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,  DOI: 10.1021/acs.jproteome.8b00155
    Google Scholar
    24
    Transcriptomic and Neuropeptidomic Analysis of the Stick Insect, Carausius morosus
    Liessem, Sander; Ragionieri, Lapo; Neupert, Susanne; Bueschges, Ansgar; Predel, Reinhard
    Journal of Proteome Research (2018), 17 (6), 2192-2204CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    One of the most thoroughly studied insect species, with respect to locomotion behavior, is the stick insect Carausius morosus. Although detailed information exists on premotor networks controlling walking, surprisingly little is known about neuropeptides, which are certainly involved in motor activity generation and modulation. So far, only few neuropeptides were identified from C. morosus or related stick insects. We performed a transcriptome anal. of the central nervous system to assemble and identify 65 neuropeptide and protein hormone precursors of C. morosus, including 5 novel putative neuropeptide precursors without clear homol. to known neuropeptide precursors of other insects (Carausius neuropeptide-like precursor 1, HanSolin, PK-like1, PK-like2, RFLamide). Using Q Exactive Orbitrap and MALDI-TOF mass spectrometry, 277 peptides including 153 likely bioactive mature neuropeptides were confirmed. Peptidomics yielded a complete coverage for many of the neuropeptide propeptides and confirmed a surprisingly high no. of heterozygous sequences. Few neuropeptide precursors commonly occurring in insects, including those of insect kinins and sulfakinins, could neither be found in the transcriptome data nor did peptidomics support their presence. The results of our study represent 1 of the most comprehensive peptidomic analyses on insects and provide the necessary input for subsequent expts. revealing neuropeptide function in greater detail.
  25. 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,  DOI: 10.1021/ac048329g
    Google Scholar
    25
    Direct Molecular Imaging of Lymnaea stagnalis Nervous Tissue at Subcellular Spatial Resolution by Mass Spectrometry
    Altelaar, A. F. Maarten; Van Minnen, Jan; Jimenez, Connie R.; Heeren, Ron M. A.; Piersma, Sander R.
    Analytical Chemistry (2005), 77 (3), 735-741CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    The imaging capabilities of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and MALDI-MS sample prepn. methods were combined. We used this method, named matrix-enhanced (ME) SIMS, for direct mol. imaging of nervous tissue at micrometer spatial resoln. Cryosections of the cerebral ganglia of the freshwater snail Lymnaea stagnalis were placed on indium-tin-oxide (ITO)-coated conductive glass slides and covered with a thin layer of 2,5-dihydroxybenzoic acid by electrospray deposition. High-resoln. mol. ion maps of cholesterol and the neuropeptide APGWamide were constructed. APGWamide was predominantly localized in the cluster of neurons that regulate male copulation behavior of Lymnaea. ME-SIMS imaging allows direct mol.-specific imaging from tissue sections without labeling and opens a complementary mass window (<2500 Da) to MALDI imaging mass spectrometry at an order of magnitude higher spatial resoln. (<3 μm).
  26. 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,  DOI: 10.1002/jms.505
    Google Scholar
    26
    Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: Practical aspects of sample preparation
    Schwartz, Sarah A.; Reyzer, Michelle L.; Caprioli, Richard M.
    Journal of Mass Spectrometry (2003), 38 (7), 699-708CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)
    Practical guidelines for the prepn. of tissue sections for direct anal. by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry are presented. Techniques for proper sample handling including tissue storage, sectioning and mounting are described. Emphasis is placed on optimizing matrix parameters such as the type of matrix mol. used, matrix concn., and solvent compn. Several different techniques for matrix application are illustrated. Optimal instrument parameters and the necessity for advanced data anal. approaches with regards to direct tissue anal. are also discussed.
  27. 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,  DOI: 10.1016/j.jasms.2008.03.016
    Google Scholar
    27
    Enhancement of Protein Sensitivity for MALDI Imaging Mass Spectrometry After Chemical Treatment of Tissue Sections
    Seeley, Erin H.; Oppenheimer, Stacey R.; Mi, Deming; Chaurand, Pierre; Caprioli, Richard M.
    Journal of the American Society for Mass Spectrometry (2008), 19 (8), 1069-1077CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
    MALDI imaging mass spectrometry (IMS) has become a valuable tool for the investigation of the content and distribution of mol. species in tissue specimens. Numerous methodol. improvements have been made to optimize tissue section prepn. and matrix deposition protocols, as well as MS data acquisition and processing. In particular for proteomic analyses, washing the tissue sections before matrix deposition has proven useful to improve spectral qualities by increasing ion yields and the no. of signals obsd. The authors systematically explore here the effects of several solvent combinations for washing tissue sections. To minimize exptl. variability, all of the measurements were performed on serial sections cut from a single mouse liver tissue block. Several other key steps of the process such as matrix deposition and MS data acquisition and processing have also been automated or standardized. To assess efficacy, after each washing procedure the total ion current and no. of peaks were counted from the resulting protein profiles. These results were correlated to on-tissue measurements obtained for lipids. Using similar approaches, several selected washing procedures were also tested for their ability to extend the lifetime as well as revive previously cut tissue sections. The effects of these washes on automated matrix deposition and crystn. behavior as well as their ability to preserve tissue histol. were also studied. Finally, in a full-scale IMS study, these washing procedures were tested on a human renal cell carcinoma biopsy.
  28. 28
    Meding, S.; Walch, A. MALDI Imaging Mass Spectrometry for Direct Tissue Analysis. Methods Mol. Biol. 2012, 931, 537– 546,  DOI: 10.1007/978-1-62703-056-4_29
    Google Scholar
    There is no corresponding record for this reference.
  29. 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,  DOI: 10.1021/acs.jproteome.8b00098
    Google Scholar
    29
    MALDI Imaging Analysis of Neuropeptides in Africanized Honeybee (Apis mellifera) Brain: Effect of Aggressiveness
    Pratavieira, Marcel; Menegasso, Anally Ribeiro da Silva; Esteves, Franciele Grego; Sato, Kenny Umino; Malaspina, Osmar; Palma, Mario Sergio
    Journal of Proteome Research (2018), 17 (7), 2358-2369CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Aggressiveness in honeybees seems to be regulated by multiple genes, under the influence of different factors, such as polyethism of workers, environmental factors, and response to alarm pheromones, creating a series of behavioral responses. It is suspected that neuropeptides seem to be involved with the regulation of the aggressive behavior. The role of allatostatin and tachykinin-related neuropeptides in honeybee brain during the aggressive behavior is unknown, and thus worker honeybees were stimulated to attack and to sting leather targets hung in front of the colonies. The aggressive individuals were collected and immediately frozen in liq. nitrogen; the heads were removed and sliced at sagittal plan. The brain slices were submitted to MALDI spectral imaging anal., and the results of the present study reported the processing of the precursors proteins into mature forms of the neuropeptides AmAST A (59-76) (AYTYVSEYKRLPVYNFGL-NH2), AmAST A (69-76) (LPVYNFGL-NH2), AmTRP (88-96) (APMGFQGMR-NH2), and AmTRP (254-262) (ARMGFHGMR-NH2), which apparently acted in different neuropils of the honeybee brain during the aggressive behavior, possibly taking part in the neuromodulation of different aspects of this complex behavior. These results were biol. validated by performing aggressiveness-related behavioral assays using young honeybee workers that received 1 ng of AmAST A (69-76) or AmTRP (88-96) via hemocele. The young workers that were not expected to be aggressive individuals presented a complete series of aggressive behaviors in the presence of the neuropeptides, corroborating the hypothesis that correlates the presence of mature AmASTs A and AmTRPs in the honeybee brain with the aggressiveness of this insect.
  30. 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,  DOI: 10.1021/ac9507956
    Google Scholar
    30
    Influence of Matrix Solution Conditions on the MALDI-MS Analysis of Peptides and Proteins
    Cohen, Steven L.; Chait, Brian T.
    Analytical Chemistry (1996), 68 (1), 31-7CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Sample-matrix prepn. procedures are shown to greatly influence the quality of the matrix-assisted laser desorption/ionization (MALDI) mass spectra of peptides and proteins. In particular, dramatic mass discrimination effects are obsd. when the matrix 4-hydroxy-α-cyanocinnamic acid is used for analyzing complex mixts. of peptides and proteins. The discrimination effects are strongly dependent on the sample-matrix soln. compn., pH, and the rates at which the sample-matrix cocrystals are grown. These findings demonstrate the need to exercise great care in performing and interpreting the MALDI anal. of biol. samples. The results also indicate that there is a reverse-phase chromatog.-like dimension in the sample-matrix prepn. procedures that can be exploited to optimize the anal. The present work describes the conditions under which the majority of components of a complex mixt. of peptides and proteins can be successfully measured.
  31. 31
    Ong, T.-H.; Romanova, E. V.; Roberts-Galbraith, R. H.; Yang, N.; 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,  DOI: 10.1074/jbc.M115.709196
    Google Scholar
    There is no corresponding record for this reference.
  32. 32
    Sadeghi, M.; Vertes, A. Crystallite Size Dependence of Volatilization in Matrix-Assisted Laser Desorption Ionization. Appl. Surf. Sci. 1998, 127–129, 226– 234,  DOI: 10.1016/S0169-4332(97)00636-3
    Google Scholar
    32
    Crystallite size dependence of volatilization in matrix-assisted laser desorption ionization
    Sadeghi, Mehrnoosh; Vertes, Akos
    Applied Surface Science (1998), 127-129 (), 226-234CODEN: ASUSEE; ISSN:0169-4332. (Elsevier Science B.V.)
    During the desorption and ionization processes in matrix-assisted laser desorption ionization (MALDI), the laser beam interacts with different areas of the polycryst. sample surface varying in crystal size and structure. CCD imaging of dried-droplet and electrospray deposited MALDI samples was used to explore the effect of laser exposure on surface morphol. at atm. pressure. Crystal size distributions of the sinapinic acid target were examd. prior to and after laser irradn. Image processing and anal. of the difference images revealed the morphol. changes in the sample. Near-threshold irradiance smaller crystals (∼2 μm) are completely volatilized by the laser shot, whereas larger crystals undergo layer-by-layer evapn. (peeling). Heat conduction simulations in a finite slab demonstrated that under similar conditions, the surface temp. of small crystallites increases markedly compared to their larger counterparts. These higher surface temps. can lead to the selective volatilization of smaller crystallites. This study points to the influence of sample prepn. on the crystal size distribution and its consequence on volatilization in MALDI-mass spectrometry (MS).
  33. 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,  DOI: 10.1007/s00216-014-8305-7
    Google Scholar
    33
    Towards imaging metabolic pathways in tissues
    Dekker, Tim J. A.; Jones, Emrys A.; Corver, Willem E.; van Zeijl, Rene J. M.; Deelder, Andre M.; Tollenaar, Rob A. E. M.; Mesker, Wilma E.; Morreau, Hans; McDonnell, Liam A.
    Analytical and Bioanalytical Chemistry (2015), 407 (8), 2167-2176CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging using 9-aminoacridine as the matrix leads to the detection of low mass metabolites and lipids directly from cancer tissues. These included lactate and pyruvate for studying the Warburg effect, as well as succinate and fumarate, metabolites whose accumulation is assocd. with specific syndromes. By using the pathway information present in the human metabolome database, it was possible to identify regions within tumor tissue samples with distinct metabolic signatures that were consistent with known tumor biol. The authors present a data anal. workflow for assessing metabolic pathways in their histopathol. context.
  34. 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. Multimodal Mass Spectrometry Imaging of N-Glycans and Proteins from the Same Tissue Section. Anal. Chem. 2016, 88, 7745– 7753,  DOI: 10.1021/acs.analchem.6b01739
    Google Scholar
    34
    Multimodal Mass Spectrometry Imaging of N-Glycans and Proteins from the Same Tissue Section
    Heijs, Bram; Holst, Stephanie; Briaire-de Bruijn, Inge H.; van Pelt, Gabi W.; de Ru, Arnoud H.; van Veelen, Peter A.; Drake, Richard R.; Mehta, Anand S.; Mesker, Wilma E.; Tollenaar, Rob A.; Bovee, Judith V. M. G.; Wuhrer, Manfred; McDonnell, Liam A.
    Analytical Chemistry (Washington, DC, United States) (2016), 88 (15), 7745-7753CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated mol. information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the in situ multimodal anal. of N-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major anal. aspects, such as lateral diffusion of the analyte mols. and differences in measurement sensitivity due to the addnl. sample prepn. methods, have been investigated for both N-glycans and proteolytic peptides. By combining the MSI approach with ext. anal., we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique N-glycan and peptide identities.
  35. 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, 208, 283– 297,  DOI: 10.1007/BF00234877
    Google Scholar
    35
    Identification of neuronal pathways between the stomatogastric nervous system and the retrocerebral complex of the cockroach Periplaneta americana (L.)
    Gundel M; Penzlin H
    Cell and tissue research (1980), 208 (2), 283-97 ISSN:0302-766X.
    The neuronal pathways connecting the stomatogastric nervous system with the retrocerebral complex of the cockroach, Periplaneta americana, were investigated by means of axonal cobalt chloride iontophoresis. Somata in the hypocerebral ganglion and in the nervus recurrens sending their axons to different parts of the stomatogastric nervous system were traced. Some axons in the oesophageal nerve arise from large perikarya in the anterior part of the pars intercerebralis and pass via the NCCI to the corpora cardiaca and the oesophageal nerve. The form a profuse dendritic tree in the protocerebrum. Fibers of the NCC I and NCC II as well as the NCA I and NCA II enter the stomatogastric nervous system via the hypocerebral ganglion.
  36. 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,  DOI: 10.1016/0020-7322(84)90033-3
    Google Scholar
    There is no corresponding record for this reference.
  37. 37
    Predel, R. Peptidergic Neurohemal System of an Insect: Mass Spectrometric Morphology. J. Comp. Neurol. 2001, 436, 363– 375,  DOI: 10.1002/cne.1073
    Google Scholar
    37
    Peptidergic neurohemal system of an insect: Mass spectrometric morphology
    Predel, Reinhard
    Journal of Comparative Neurology (2001), 436 (3), 363-375CODEN: JCNEAM; ISSN:0021-9967. (Wiley-Liss, Inc.)
    Neuropeptides are by far the most diverse group of messenger mols. in insects. To understand cell signaling and function, it is essential to reveal the complete neuropeptide profile of a single neuron/nerve/neurohemal organ first. In this study, matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry was used to analyze the peptidergic system of an insect, focusing on the neurohemal structures. Major neurohemal organs were investigated, including the retrocerebral complex, perisympathetic organs, and all nerves supplying these organs with neurosecretions. Addnl., peripheral neurohemal release sites such as the dilator muscle of the antennal circulatory organ and lateral heart nerves were studied, as well as parts of the stomatogastric nervous system. The following neuropeptide families were analyzed: kinins, allatostatins, leucomyosuppressin, corazonin, adipokinetic hormones, myoinhibitory peptide, sulfakinins, periviscerokinins, YLSamide, VEAacid, SKNacid, proctolin, the head peptide, and pyrokinins. Beyond a contribution to a map of the distribution of neuropeptides in a neurohemal system, the following conclusions can be drawn from these expts. (1) Nearly all abundant peaks in the different mass spectra represent peptides that have already been identified. (2) Although only adult males were used in this study, variations in the peptide abundances were obsd. that are possibly correlated with different physiol./developmental conditions. (3) Peptides have a body-region-specific distribution in the neurohemal system. (4) A clear compartmentalization of the retrocerebral complex could be obsd.
  38. 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,  DOI: 10.1002/cne.903220105
    Google Scholar
    38
    Neurons in the cockroach nervous system reacting with antisera to the neuropeptide leucokinin I
    Nassel D R; Cantera R; Karlsson A
    The Journal of comparative neurology (1992), 322 (1), 45-67 ISSN:0021-9967.
    Antisera were raised against the myotropic neuropeptide leucokinin I, originally isolated from head extracts of the cockroach Leucophaea maderae. Processes of leucokinin I immunoreactive (LKIR) neurons were distributed throughout the nervous system, but immunoreactive cell bodies were not found in all neuromeres. In the brain, about 160 LKIR cell bodies were distributed in the protocerebrum and optic lobes (no LKIR cell bodies were found in the deuto- and tritocerebrum). In the ventral ganglia, LKIR cell bodies were seen distributed as follows: eight (weakly immunoreactive) in the subesophageal ganglion; about six larger and bilateral clusters of 5 smaller in each of the three thoracic ganglia, and in each of the abdominal ganglia, two pairs of strongly immunoreactive cell bodies were resolved. Many of the LKIR neurons could be described in detail. In the optic lobes, immunoreactive neurons innervate the medulla and accessory medulla. In the brain, three pairs of bilateral LKIR neurons supply branches to distinct sets of nonglomerular neuropil, and two pairs of descending neurons connect the posterior protocerebrum to the antennal lobes and all the ventral ganglia. Other brain neurons innervate the central body, tritocerebrum, and nonglomerular neuropil in protocerebrum. LKIR neurons of the median and lateral neurosecretory cell groups send axons to the corpora cardiaca, frontal ganglion, and tritocerebrum. In the muscle layer of the foregut (crop), bi- and multipolar LKIR neurons with axons running to the retrocerebral complex were resolved. The LKIR neurons in the abdominal ganglia form efferent axons supplying the lateral cardiac nerves, spiracles, and the segmental perivisceral organs. The distribution of immunoreactivity indicates roles for leucokinins as neuromodulators or neurotransmitters in central interneurons arborizing in different portions of the brain, visual system, and ventral ganglia. Also, a function in circuits regulating feeding can be presumed. Furthermore, a role in regulation of heart and possibly respiration can be suggested, and probably leucokinins are released from corpora cardiaca as neurohormones. Leucokinins were isolated by their myotropic action on the Leucophaea hindgut, but no innervation of this portion of the gut could be demonstrated. The distribution of leucokinin immunoreactivity was compared to immunolabeling with antisera against vertebrate tachykinins and lysine vasopressin.
  39. 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,  DOI: 10.1021/acs.jproteome.7b00630
    Google Scholar
    39
    Neuropeptidomics of the Bed Bug Cimex lectularius
    Predel, Reinhard; Neupert, Susanne; Derst, Christian; Reinhardt, Klaus; Wegener, Christian
    Journal of Proteome Research (2018), 17 (1), 440-454CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    The bed bug Cimex lectularius is a globally distributed human ectoparasite with fascinating biol. It has recently acquired resistance against a broad range of insecticides, causing a worldwide increase in bed bug infestations. The recent annotation of the bed bug genome revealed a full complement of neuropeptide and neuropeptide receptor genes in this species. With regard to the biol. of C. lectularius, neuropeptide signaling is esp. interesting because it regulates feeding, diuresis, digestion, as well as reprodn. and also provides potential new targets for chem. control. To identify which neuropeptides are translated from the genome-predicted genes, we performed a comprehensive peptidomic anal. of the central nervous system of the bed bug. We identified in total 144 different peptides from 29 precursors, of which at least 67 likely present bioactive mature neuropeptides. C. lectularius corazonin and myosuppressin are unique and deviate considerably from the canonical insect consensus sequences. Several identified neuropeptides likely act as hormones, as evidenced by the occurrence of resp. mass signals and immunoreactivity in neurohemal structures. Our data provide the most comprehensive peptidome of a Heteropteran species so far and in comparison suggest that a hematophageous life style does not require qual. adaptations of the insect peptidome.
  40. 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,  DOI: 10.1021/pr5011636
    Google Scholar
    40
    Neuropeptidomics of the Carpenter Ant Camponotus floridanus
    Schmitt, Franziska; Vanselow, Jens T.; Schlosser, Andreas; Kahnt, Joerg; Roessler, Wolfgang; Wegener, Christian
    Journal of Proteome Research (2015), 14 (3), 1504-1514CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Ants show a rich behavioral repertoire and a highly complex organization, which have been attracting behavioral and sociobiol. researchers for a long time. The neuronal underpinnings of ant behavior and social organization are, however, much less understood. Neuropeptides are key signals that orchestrate animal behavior and physiol., and it is thus feasible to assume that they play an important role also for the social constitution of ants. Despite the availability of different ant genomes and in silico prediction of ant neuropeptides, a comprehensive biochem. survey of the neuropeptidergic communication possibilities of ants is missing. We therefore combined different mass spectrometric methods to characterize the neuropeptidome of the adult carpenter ant Camponotus floridanus. We also characterized the local neuropeptide complement in different parts of the nervous and neuroendocrine system, including the antennal and optic lobes. Our anal. identifies 39 neuropeptides encoded by different prepropeptide genes, and in silico predicts new prepropeptide genes encoding CAPA peptides, CNMamide as well as homologs of the honey bee IDLSRFYGHFNT- and ITGQGNRIF-contg. peptides. Our data provides basic information about the identity and localization of neuropeptides that is required to anatomically and functionally address the role and significance of neuropeptides in ant behavior and physiol.
  41. 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. 2004, 88, 311– 319,  DOI: 10.1046/j.1471-4159.2003.02161.x
    Google Scholar
    41
    Expression of a novel neuropeptide, NVGTLARDFQLPIPNamide, in the larval and adult brain of Drosophila melanogaster
    Verleyen, Peter; Baggerman, Geert; Wiehart, Ursula; Schoeters, Eric; Van Lommel, Alfons; De Loof, Arnold; Schoofs, Liliane
    Journal of Neurochemistry (2004), 88 (2), 311-319CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)
    Advances in mass spectrometry and the availability of genomic databases made it possible to det. the peptidome or peptide content of a specific tissue. Peptidomics by nanoflow capillary liq. chromatog. tandem mass spectrometry of an ext. of 50 larval Drosophila brains, yielded 28 neuropeptides. Eight were entirely novel and encoded by five not yet annotated genes; only two genes had a homolog in the Anopheles gambiae genome. Seven of the eight peptides did not show relevant sequence homol. to any known peptide. Therefore, no evidence towards the physiol. role of these "orphan" peptides was available. We identified one of the eight peptides, IPNamide, in an ext. of the Drosophila adult brain as well. Next, specific antisera were raised to reveal the distribution pattern of IPNamide and other peptides from the same precursor, in larval and adult brains by means of whole-mount immunocytochem. and confocal microscopy. IPNamide immunoreactivity is abundantly present in both stages and a striking similarity was found between the distribution patterns of IPNamide and TPAEDFMRFamide, a member of the FMRFamide peptide family. Based on this distribution pattern, IPNamide might be involved in phototransduction, in processing sensory stimuli, as well as in controlling the activity of the esophagus.
  42. 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-Enkephalin-Arg6-Gly7-Leu8. Cell Tissue Res. 1991, 263, 285– 291,  DOI: 10.1007/BF00318770
    Google Scholar
    42
    Immunocytochemical mapping of neuronal pathways from brain to corpora cardiaca/corpora allata in the cockroach Diploptera punctata with antisera against Met-enkephalin-Arg6-Gly7-Leu8
    Duve H; Thorpe A; Tobe S S
    Cell and tissue research (1991), 263 (2), 285-91 ISSN:0302-766X.
    Neuronal circuits in the brain and retrocerebral complex of the cockroach Diploptera punctata have been mapped immunocytochemically with antisera directed against the extended enkephalin, Met-enkephalin-Arg6-Gly7-Leu8 (Met-8). The pathways link median and lateral neurosecretory cells with the corpus cardiacum corpus allatum complex. In females, nerve fibres penetrate the corpora allata and varicosities or terminals, immunoreactive to Met-8, surround the glandular cells. Males differ in having almost no Met-8 immunoreactivity in the corpora allata. The corpora cardiaca of both males and females are richly supplied with Met-8 immunoreactive material, in particular in the 'cap' regions immediately adjacent to the corpora allata. A similarity in the amino-acid sequences of Met-8 and the C-terminus of the recently characterised allatostatins of D. punctata suggests that the pathways identified with the Met-8 antisera may be the same as those by which the allatostatins are transported from the brain to the corpus allatum. In comparative studies on the blowfly Calliphora vomitoria, similar neuronal pathways have been identified except that no sexual dimorphism with respect to amounts of immunoreactive material within the corpus allatum has been observed. These results suggest a possible homology in the neuropeptide regulation of the gland.
  43. 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,  DOI: 10.1007/BF00327985
    Google Scholar
    43
    Localization of corazonin in the nervous system of the cockroach Periplaneta americana
    Veenstra, Jan A.; Davis, Norman T.
    Cell & Tissue Research (1993), 274 (1), 57-64CODEN: CTSRCS; ISSN:0302-766X.
    Antisera raised to the cardioactive peptide corazonin were used to localize immunoreactive cells in the nervous system of the American cockroach. Sera obtained after the seventh booster injection were sufficiently specific to be used for immunocytol. They recognized a subset of 10 lateral neurosecretory cells in the proto-cerebrum that project to, and arborize and terminate in the ipsilateral corpus cardiacum. They also reacted with bilateral neurons in each of the thoracic and abdominal neuromeres, a single dorsal unpaired median neuron in the subesophageal ganglion, an interneuron in each optic lobe, and other neurons at the base of the optic lobe, in the tritocerebrum and deutocerebrum. The presence of corazonin in the abdominal neurons and the lateral neurosecretory cells was confirmed by HPLC fractionation of exts. of the abdominal ganglia, brains and retrocerebral complexes, followed by detn. of corazonin by ELISA, which revealed in each tissue a single immunoreactive peak co-eluting with corazonin in two different HPLC systems. Antisera obtained after the first three booster injections recognized a large no. of neuroendocrine cells and neurons in the brain and the abdominal nerve cord. However, the sera from the two rabbits reacted largely with different cells, indicating that the majority of this immunoreactivity was due to cross-reactivity. These results indicate that the prodn. of highly specific antisera to some neuropeptides may require a considerable no. of booster injections.
  44. 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,  DOI: 10.1002/cne.21183
    Google Scholar
    44
    Peptidomics of identified neurons demonstrates a highly differentiated expression pattern of FXPRLamides in the neuroendocrine system of an insect
    Predel Reinhard; Eckert Manfred; Pollak Edit; Molnar Laszlo; Scheibner Olaf; Neupert Susanne
    The Journal of comparative neurology (2007), 500 (3), 498-512 ISSN:0021-9967.
    FXPRLamides are insect neuropeptides that mediate such diverse functions as pheromone biosynthesis, visceral muscle contraction, and induction of diapause. Although multiple forms occur in every insect studied so far, little is known about a possible functional differentiation and/or differences in the cellular expression pattern of these messenger molecules. In this study, we performed a mass spectrometric survey of all FXPRLamide-expressing neurosecretory neurons in the CNS of Periplaneta americana. That species combines a very well characterized peptidergic system with relatively easy accessible neurosecretory cells suitable for dissection. In addition to the extensive mass spectrometric analyses of single cells, the projection of the FXPRLamide-expressing neurons was studied with three antisera specifically recognizing different FXPRLamides. The following conclusions can be drawn from this first comprehensive peptidomic approach on insect neurons. 1) A high degree of differentiation in the expression of FXPRLamides exists; not fewer then four cell types containing different sets of FXPRLamides were observed. 2) A low level of colocalization with other neuropeptides was found in these neurons. 3) A comparison with FXPRLamide-expressing neurons of other insects shows a high degree of conservation in the localization and projection of these neurons, which is not corroborated by a similar conservation of the corresponding peptide sequences. 4) Although the methods for cell identification, dissection, and sample preparation for mass spectrometry were kept as simple as possible, it was unambiguously shown that this approach is generally suitable for routine analysis of single identified neurons of insects.
  45. 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,  DOI: 10.1016/j.jasms.2007.06.010
    Google Scholar
    45
    Sublimation as a Method of Matrix Application for Mass Spectrometric Imaging
    Hankin, Joseph A.; Barkley, Robert M.; Murphy, Robert C.
    Journal of the American Society for Mass Spectrometry (2007), 18 (9), 1646-1652CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
    Common org. matrix-assisted laser desorption/ionization (MALDI) matrixes, 2,5-dihydroxybenzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, and α-cyano-4-hydroxycinnamic acid, were found to undergo sublimation without decompn. under conditions of reduced pressure and elevated temp. This solid to vapor-phase transition was exploited to apply MALDI matrix onto tissue samples over a broad surface in a solvent-free application for mass spectrometric imaging. Sublimation of matrix produced an even layer of small crystals across the sample plate. The deposition was readily controlled with time, temp., and pressure settings and was highly reproducible from one sample to the next. Mass spectrometric images acquired from phospholipid stds. robotically spotted onto a MALDI plate yielded a more intense, even signal with fewer sodium adducts when matrix was applied by sublimation relative to samples where matrix was deposited by an electrospray technique. MALDI matrix could be readily applied to tissue sections on glass slides and stainless steel MALDI plate inserts as long as good thermal contact was made with the condenser of the sublimation device. Sections of mouse brain were coated with matrix applied by sublimation and were imaged using a Q-q-TOF mass spectrometer to yield mass spectral images of very high quality. Image quality is likely enhanced by several features of this technique including the microcryst. morphol. of the deposited matrix, increased purity of deposited matrix, and evenness of deposition. This inexpensive method was reproducible and eliminated the potential for spreading of analytes arising from solvent deposition during matrix application.
  46. 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,  DOI: 10.1021/ac200998a
    Google Scholar
    46
    Matrix Sublimation/Recrystallization for Imaging Proteins by Mass Spectrometry at High Spatial Resolution
    Yang, Junhai; Caprioli, Richard M.
    Analytical Chemistry (Washington, DC, United States) (2011), 83 (14), 5728-5734CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    We have employed matrix deposition by sublimation for protein image anal. on tissue sections using a hydration/recrystn. process that produces high-quality MALDI mass spectra and high-spatial-resoln. ion images. We systematically investigated different washing protocols, the effect of tissue section thickness, the amt. of sublimated matrix per unit area, and different recrystn. conditions. The results show that an org. solvent rinse followed by ethanol/water rinses substantially increased sensitivity for the detection of proteins. Both the thickness of the tissue section and the amt. of sinapinic acid sublimated per unit area have optimal ranges for maximal protein signal intensity. Ion images of mouse and rat brain sections at 50, 20, and 10 μm spatial resoln. are presented and are correlated with hematoxylin and eosin (H&E)-stained optical images. For targeted anal., histol.-directed imaging can be performed using this protocol where MS anal. and H&E staining are performed on the same section.
  47. 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,  DOI: 10.1038/nmeth.4433
    Google Scholar
    47
    Autofocusing MALDI mass spectrometry imaging of tissue sections and 3D chemical topography of nonflat surfaces
    Kompauer, Mario; Heiles, Sven; Spengler, Bernhard
    Nature Methods (2017), 14 (12), 1156-1158CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
    We describe an atm. pressure matrix-assisted laser desorption-ionization mass spectrometry imaging system that uses long-distance laser triangulation on a micrometer scale to simultaneously obtain topog. and mol. information from 3D surfaces. We studied the topog. distribution of compds. on irregular 3D surfaces of plants and parasites, and we imaged nonplanar tissue sections with high lateral resoln., thereby eliminating height-related signal artifacts.

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  • Abstract

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    Figure 1

    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 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.

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    Figure 2

    Figure 2. FMRF paracopies in mass spectra from RCC preparations. (A) MSI from a single tissue section showing the distributions of four FMRFs, suggesting identical spatial distributions of these peptides in the RCC. Section: 20 μm, 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 an RCC extract spotted on an ITO glass slide. The matrix-spraying and MALDI-TOF equipment were exactly the same as those as used for (B). The accuracy of mass matching for peptide assignment was settled at ±0.25 Da.

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    Figure 3

    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 an overview of the architecture of RCC–SNS). Scale bar (white): 600 μm, section thicknesses: (A–I) 20 μm and (J–L) 14 μm.

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    Figure 4

    Figure 4. Distribution of corazonin and AstA analyzed in serial RCC sections by (A) immunohistochemistry and (B) MSI (the more peripheral section). Data obtained by both methods confirmed the different spatial distributions 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%. The accuracy of mass matching for peptide assignment was settled at ±0.25 Da.

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    Figure 5

    Figure 5. (A) Ion maps of four PKs indicating differential processing of the PK precursor. (B) Four PKs detected in the posterior part of the RCC, which mostly contains PKs processed in cells of the SEG. (C) Anterior corpus cardiacum tissue, which receives neuropeptides from the brain, showing no 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%). The accuracies of mass matching for peptide assignment were settled at ±0.25 Da for PK-2, -3, and -4 and at ±0.001 Da for PK-1. Tissue sections were not washed with ethanol prior to matrix spraying.

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    Figure 6

    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) and the glandular and neurohemal corpora cardiaca (CC). The neurohemal part of the corpora cardiaca is further subdivided into three subcompartments.

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  • References

    This article references 47 other publications.

    1. 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,  DOI: 10.1016/j.bbrc.2005.07.038
      1
      The Drosophila gene CG9918 codes for a pyrokinin-1 receptor
      Cazzamali, Giuseppe; Torp, Malene; Hauser, Frank; Williamson, Michael; Grimmelikhuijzen, Cornelis J. P.
      Biochemical and Biophysical Research Communications (2005), 335 (1), 14-19CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)
      The database from the Drosophila Genome Project contains a gene, CG9918, annotated to code for a G protein-coupled receptor. We cloned the cDNA of this gene and functionally expressed it in Chinese hamster ovary cells. We tested a library of about 25 Drosophila and other insect neuropeptides, and seven insect biogenic amines on the expressed receptor and found that it was activated by low concns. of the Drosophila neuropeptide, pyrokinin-1 (TGPSASSGLWFGPRLamide; EC50, 5 × 10-8 M). The receptor was also activated by other Drosophila neuropeptides, terminating with the sequence PRLamide (Hug-γ, ecdysis-triggering-hormone-1, pyrokinin-2), but in these cases about six to eight times higher concns. were needed. The receptor was not activated by Drosophila neuropeptides, contg. a C-terminal PRIamide sequence (such as ecdysis-triggering-hormone-2), or PRVamide (such as capa-1 and -2), or other neuropeptides and biogenic amines not related to the pyrokinins. This paper is the first conclusive report that CG9918 is a Drosophila pyrokinin-1 receptor gene.
    2. 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,  DOI: 10.1016/S0006-291X(02)02709-2
      There is no corresponding record for this reference.
    3. 3
      Smith, M. E. POMC Opioid Peptides. In Handbook of Biologically Active Peptides; Kastin, A. J., Ed.; Elsevier: San Diego, 2006; pp 1325– 1331.
      There is no corresponding record for this reference.
    4. 4
      Rubakhin, S. S.; Sweedler, J. V. Characterizing Peptides in Individual Mammalian Cells Using Mass Spectrometry. Nat. Protoc. 2007, 2, 1987– 97,  DOI: 10.1038/nprot.2007.277
      4
      Characterizing peptides in individual mammalian cells using mass spectrometry
      Rubakhin, Stanislav S.; Sweedler, Jonathan V.
      Nature Protocols (2007), 2 (8), 1987-1997CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
      Cell-to-cell chem. signaling plays multiple roles in coordinating the activity of the functional elements of an organism, with these elements ranging from a three-neuron reflex circuit to the entire animal. In recent years, single-cell mass spectrometry (MS) has enabled the discovery of cell-to-cell signaling mols. from the nervous system of a no. of invertebrates. The authors describe a protocol for analyzing individual cells from rat pituitary using matrix-assisted laser desorption/ionization MS. Each step in the sample prepn. process, including cell stabilization, isolation, sample prepn., signal acquisition and data interpretation, is detailed here. Although the authors employ this method to investigate peptides in individual pituitary cells, it can be adapted to other cell types and even subcellular sections from a range of animals. This protocol allows one to obtain 20-30 individual cell samples and acquire mass spectra from them in a single day.
    5. 5
      Neupert, S.; Johard, H. A. D.; Nässel, D. R.; Predel, R. Single-Cell Peptidomics of Drosophila melanogaster Neurons Identified by Gal4-Driven Fluorescence. Anal. Chem. 2007, 79, 3690– 3694,  DOI: 10.1021/ac062411p
      5
      Single-Cell Peptidomics of Drosophila melanogaster Neurons Identified by Gal4-Driven Fluorescence
      Neupert, Susanne; Johard, Helena A. D.; Naessel, Dick R.; Predel, Reinhard
      Analytical Chemistry (Washington, DC, United States) (2007), 79 (10), 3690-3694CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Neuropeptides are widespread signal mols. that display a great chem. and functional diversity. Predictions of neuropeptide cleavage from precursor proteins are not always correct, and thus, biochem. identification is essential. Single-cell anal. is valuable to identify peptides processed from a single precursor, but also to det. coexpression of further neuropeptides from other precursors. The authors have developed an approach to isolate single identified neurons from the fruit fly Drosophila melanogaster for mass spectrometric anal. By using Gal4 promoter lines to drive green fluorescent protein under UAS control, the authors identified specific peptidergic neurons. These neurons were isolated singly under a fluorescence microscope and subjected to MALDI-TOF mass spectrometry. Two Gal4 lines were used here to identify pigment-dispersing factor (PDF) and hugin-expressing neurons. The authors found that the large PDF expressing clock neurons only give rise to a single peptide, PDF. The three different classes of hugin expressing neurons all display the same mass signal, identical to pyrokinin-2. The other peptide predicted from the hugin precursor, hugin γ, was not detected in any of the cells. Single-cell peptidomics is a powerful tool in Drosophila neuroscience since Gal4 drivers can be produced for all known neuropeptide genes and thus provide detailed information about neuropeptide complements in neurons of interest.
    6. 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,  DOI: 10.1007/s00216-010-3723-7
      6
      Mass spectral imaging and profiling of neuropeptides at the organ and cellular domains
      Chen, Ruibing; Li, Lingjun
      Analytical and Bioanalytical Chemistry (2010), 397 (8), 3185-3193CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      A review. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a rapid and sensitive anal. method that is well suited for detg. mol. wts. of peptides and proteins from complex samples. MALDI-MS can be used to profile the peptides and proteins from single-cell and small tissue samples without the need for extensive sample prepn. Furthermore, the recently developed MALDI imaging technique enables mapping of the spatial distribution of signaling mols. in tissue samples. Several examples of signaling mol. anal. at the single-cell and single-organ levels using MALDI-MS technol. are highlighted followed by an outlook of future directions.
    7. 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,  DOI: 10.1021/acs.analchem.7b04733
      7
      Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights
      Buchberger, Amanda Rae; DeLaney, Kellen; Johnson, Jillian; Li, Lingjun
      Analytical Chemistry (Washington, DC, United States) (2018), 90 (1), 240-265CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A review. Mass spectrometry imaging (MSI) is a powerful tool that enables untargeted studies into the spatial distribution of mol. species in a variety of samples. It has the capability to image thousands of mols., such as metabolites, lipids, peptides, proteins, and glycans, in a single expt. without labeling. The combination of information gained from mass spectrometry (MS) and visualization of spatial distributions in thin sample sections makes this a valuable chem. anal. tool useful for biol. specimen characterization. After minimal but careful sample prepn., the general setup of an MSI expt. involves defining an (x, y) grid over the surface of the sample, with the grid area chosen by the user. The mass spectrometer then ionizes the mols. on the surface of the sample and collects a mass spectrum at each pixel on the section, with the resulting spatial resoln. defined by the pixel size. After collecting the spectra, computational software can be used to select an individual mass-to-charge (m/z) value, and the intensity of the m/z is extd. from each pixel's spectrum. These intensities are then combined into a heat map image depicting the relative distribution of that m/z value throughout the sample's surface. In order to det. the identity of a specific m/z value, tandem MS (MS/MS) fragmentation can be performed on ions from each pixel, and the fragments can be used to piece together the structure of the unknown mol. Otherwise, the mol. can be identified based on its intact mass by accurate mass matching to databases of known mols. within a certain mass error range. Overall, the aim of this review is to provide an informative resource for those in the MSI community who are interested in improving MSI data quality and anal. or using MSI for novel applications. Particularly, advances from the last two years in sample prepn., instrumentation, quantitation, statistics, and multi-modal imaging that have allowed MSI to emerge as a powerful technique in various biomedical applications including clin. settings are discussed. Also, several novel biol. applications are highlighted to demonstrate the potential for the future of the MSI field.
    8. 8
      Spengler, B. Mass Spectrometry Imaging of Biomolecular Information. Anal. Chem. 2015, 87, 64– 82,  DOI: 10.1021/ac504543v
      8
      Mass Spectrometry Imaging of Biomolecular Information
      Spengler, Bernhard
      Analytical Chemistry (Washington, DC, United States) (2015), 87 (1), 64-82CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A review. Mass spectrometry imaging (MSI) has gone through a significant change over the last three years, the time span of this review. The predominant mass spectrometric method in biomol. imaging has been matrix-assisted laser desorption/ionization (MALDI). MALDI MSI, after its first introduction in 1994 [1] has suffered from a phase of rather unsystematic use, without validation on an anal.-science level. Not meeting a high std. of validity for these colorful pictures, MSI reached a dangerous state of being suspected as a non-trustful method, useless for precise biomedical or mol.-biol. research. With the introduction of new, highly sensitive, highly accurate methods of data acquisition and imaging, which were based on high mass resoln., high mass accuracy and high lateral resoln., MALDI MSI recovered and developed as a powerful, versatile and valid method in bioanal. sciences [2-4]. At the same time alternative ionization methods came into the game, providing imaging capability with reduced sample prepn. requirements and specific anal. properties. The last three years have thus been characterized by developments and applications in biomol. imaging, which are unprecedented not only in the view of mass spectrometrists.
    9. 9
      Chen, R.; Cape, S. S.; Sturm, R. M.; Li, L. Mass Spectrometric Imaging of Neuropeptides in Decapod Crustacean Neuronal Tissues. Methods Mol. Biol. 2010, 656, 451– 463,  DOI: 10.1007/978-1-60761-746-4_26
      9
      Mass spectrometric imaging of neuropeptides in decapod crustacean neuronal tissues
      Chen, Ruibing; Cape, Stephanie S.; Sturm, Robert M.; Li, Lingjun
      Methods in Molecular Biology (New York, NY, United States) (2010), 656 (Mass Spectrometry Imaging), 451-463CODEN: MMBIED; ISSN:1064-3745. (Springer)
      The emerging technol. mass spectrometric imaging (MSI) provides an attractive opportunity to detect and probe the mol. content of tissues in an anatomical context. This powerful methodol. has been applied extensively to the localization of proteins, peptides, pharmaceuticals, metabolites, lipids, and other biol. and chem. compds. in tissues. Herein, we present a method developed specifically for mapping neuropeptides in crustacean neuronal tissues. Both cryostat tissue sectioning and whole-mount tissue blotting techniques are highlighted. Careful sample prepn. is essential for obtaining sufficient analyte/matrix mixing while retaining the spatial localization of the neuropeptides. Several matrix application app. and techniques are described and compared. Furthermore, three-dimensional (3D) imaging has been developed to provide detailed information about the distribution of neuropeptides within 3D structure of a crustacean brain.
    10. 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,  DOI: 10.1021/pr900736t
      10
      Mass Spectral Analysis of Neuropeptide Expression and Distribution in the Nervous System of the Lobster Homarus americanus
      Chen, Ruibing; Jiang, Xiaoyue; Prieto Conaway, Maria C.; Mohtashemi, Iman; Hui, Limei; Viner, Rosa; Li, Lingjun
      Journal of Proteome Research (2010), 9 (2), 818-832CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      The lobster Homarus americanus has long served as an important animal model for electrophysiol. and behavioral studies. Using this model, we performed a comprehensive investigation of the neuropeptide expression and their localization in the nervous system, which provides useful insights for further understanding of their biol. functions. Using nanoLC ESI Q-TOF MS/MS and 3 types of MALDI instruments, we analyzed the neuropeptide complements in a major neuroendocrine structure, pericardial organ. A total of 57 putative neuropeptides were identified and 18 of them were de novo sequenced. Using direct tissue/ext. anal. and bioinformatics software SpecPlot, we charted the global distribution of neuropeptides throughout the nervous system in H. americanus. Furthermore, we also mapped the localization of several neuropeptide families in the brain by high mass resoln. and high mass accuracy mass spectrometric imaging (MSI) using a MALDI LTQ Orbitrap mass spectrometer. We have also compared the utility and instrument performance of multiple mass spectrometers for neuropeptide anal. in terms of peptidome coverage, sensitivity, mass spectral resoln. and capability for de novo sequencing.
    11. 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,  DOI: 10.1021/pr500224b
      11
      MALDI Imaging Analysis of Neuropeptides in the Africanized Honeybee (Apis mellifera) Brain: Effect of Ontogeny
      Pratavieira, Marcel; da Silva Menegasso, Anally Ribeiro; Garcia, Ana Maria Caviquioli; dos Santos, Diego Simoes; Gomes, Paulo Cesar; Malaspina, Osmar; Palma, Mario Sergio
      Journal of Proteome Research (2014), 13 (6), 3054-3064CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      The occurrence and spatial distribution of the neuropeptides AmTRP-5 and AST-1 in the honeybee brain were monitored via MALDI spectral imaging according to the ontogeny of Africanized Apis mellifera. The levels of these peptides increased in the brains of 0-15 day old honeybees, and this increase was accompanied by an increase in the no. of in-hive activities performed by the nurse bees, followed by a decrease in the period from 15 to 25 days of age, in which the workers began to perform activities outside the nest (guarding and foraging). The results obtained in the present investigation suggest that AmTRP-5 acts in the upper region of both pedunculi of young workers, possibly regulating the cell cleaning and brood capping activities. Meanwhile, the localized occurrence of AmTRP-5 and AST-1 in the antennal lobes, subesophageal ganglion, upper region of the medulla, both lobula, and α- and β-lobes of both brain hemispheres in 20-25 day old workers suggest that the action of both neuropeptides in these regions may be related to their localized actions in these regions, regulating foraging and guarding activities. Thus, these neuropeptides appear to have some functions in the honeybee brain that are specifically related to the age-related division of labor.
    12. 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,  DOI: 10.1007/978-1-60761-746-4_25
      12
      Imaging of similar mass neuropeptides in neuronal tissue by enhanced resolution MALDI MS with an ion trap - Orbitrap hybrid instrument
      Verhaert, Peter D. E. M.; Pinkse, Martijn W. H.; Strupat, Kerstin; Conaway, Maria C. Prieto
      Methods in Molecular Biology (New York, NY, United States) (2010), 656 (Mass Spectrometry Imaging), 433-449CODEN: MMBIED; ISSN:1064-3745. (Springer)
      Several mass spectrometry imaging (MSI) procedures are used to localize physiol. active peptides in neuronal tissue from American cockroach (Periplaneta americana) neurosecretory organs. We report how to use this model system to assess, for the first time, the performance of the MALDI LTQ Orbitrap XL mass spectrometer to perform MSI of secretory neuropeptides. The method involves the following steps: (1) rapid dissecting of neurosecretory tissue (i.e., insect neurohemal organ) in isotonic sucrose soln.; (2) mounting the tissue on a glass slide; (3) controlled spraying of the air-dried tissue with concd. MALDI matrix soln.; (4) loading specimen into the MALDI source of a MSn system equipped with an Orbitrap analyzer; (5) setting-up MSI methods by detg. tissue areas of interest, spatial resoln., mol. mass range, and mol. mass resoln.; (6) acquiring mass spectra; (7) analyzing data using ImageQuest MSI software to generate (single or composite) images of the distribution of peptide(s) of interest; (8) confirming the identity of selected peptides by MS2 and/or MSn sequencing directly from imaged tissue sample. The results illustrate that high mass accuracy and high mass resolving power of the Orbitrap analyzer are achievable in analyses directly from tissue, such as in MSI expts. Moreover the mass spectrometric instrumentation evaluated allows for both peptide localization and peptide identification/sequencing directly from tissue.
    13. 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,  DOI: 10.1016/j.ijms.2017.04.001
      There is no corresponding record for this reference.
    14. 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. 2012, 520, 694– 716,  DOI: 10.1002/cne.22745
      14
      Toward a single-cell-based analysis of neuropeptide expression in Periplaneta americana antennal lobe neurons
      Neupert, Susanne; Fusca, Debora; Schachtner, Joachim; Kloppenburg, Peter; Predel, Reinhard
      Journal of Comparative Neurology (2012), 520 (4), 694-716CODEN: JCNEAM; ISSN:0021-9967. (Wiley-Blackwell)
      A multitude of potential neurotransmitters and neuromodulators, including peptides, have been detected in the antennal lobe (AL), the first synaptic relay of the central olfactory pathway in the insect brain. However, the functional role of neuropeptides in this system has yet to be revealed. An important prerequisite to understanding the role of neuropeptides is to match the functionally different cell types in the AL with their peptide profiles by using electrophysiol. recordings combined with immunocytochem. studies and/or single-cell mass spectrometry. The olfactory system of Periplaneta americana is particularly well suited to accomplish this goal because several physiol. distinct neuron types can be unequivocally identified. With the aim to analyze the neuropeptide inventory of the P. americana AL, this study is an essential step in this direction. First, the authors systematically analyzed different parts of the AL by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry to obtain the complete set of neuropeptides present. Altogether, 56 ion signals could be assigned to products of 10 neuropeptide genes (allatostatins A, B, C, SIFamide, allatotropin, FMRFamide-related peptides [myosuppressin, short neuropeptides F, extended FMRFamides], crustacean cardioactive peptide, tachykinin-related peptides). In a second step, a combination of immunocytochem. and mass spectrometric profiling of defined AL compartments was used to reveal the spatial distribution of neuropeptide-contg. cells. Finally, the authors demonstrated the feasibility of MALDI-TOF mass spectrometric profiling of single AL neurons, which is an important precondition for combining electrophysiol. with peptide profiling at the single-cell level. J. Comp. Neurol., 2012. © 2011 Wiley Periodicals, Inc.
    15. 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-Year-Old Formalin-Fixed, Paraffin-Embedded Tissue by Mass Spectrometry Imaging. Anal. Chem. 2018, 90, 9272– 9280,  DOI: 10.1021/acs.analchem.8b01838
      15
      Digestion-Free Analysis of Peptides from 30-year-old Formalin-Fixed, Paraffin-Embedded Tissue by Mass Spectrometry Imaging
      Paine, Martin R. L.; Ellis, Shane R.; Maloney, Dan; Heeren, Ron M. A.; Verhaert, Peter D. E. M.
      Analytical Chemistry (Washington, DC, United States) (2018), 90 (15), 9272-9280CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Formalin-fixed neuroendocrine tissues from American cockroaches (Periplaneta americana) embedded in paraffin more than 30 years ago were recently analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), to reveal the histol. localization of more than 20 peptide ions. These represented protonated, and other cationic species of, at least, 14 known neuropeptides. The characterization of peptides in such historical samples was made possible by a novel sample prepn. protocol rendering the endogenous peptides readily amenable to MSI anal. The protocol comprises brief deparaffinization steps involving xylene and ethanol, and is further devoid of conventional aq. washing, buffer incubations, or antigen retrieval steps. Endogenous secretory peptides that are typically highly sol. are therefore retained in-tissue with this protocol. The method is fully "top-down", i.e., without laborious in situ enzymic digestion that typically disturbs the detection of low-abundance endogenous peptides by MSI. Peptide identifications were supported by accurate mass, on-tissue tandem MS analyses, and by earlier MALDI-MSI results reported for freshly prepd. P. americana samples. In contrast to earlier literature accounts stating that MALDI-MSI detection of endogenous peptides is possible only in fresh or freshly frozen tissues, or exceptionally, in formalin-fixed, paraffin-embedded (FFPE) material of less than 1 yr old, the authors demonstrate that MALDI-MSI works for endogenous peptides in FFPE tissue of up to 30 years old. The findings put forward a useful method for digestion-free, high-throughput anal. of endogenous peptides from FFPE samples and offer the potential for reinvestigating archived and historically interesting FFPE material, such as those stored in hospital biobanks.
    16. 16
      Gäde, G.; Hoffmann, K. H.; Spring, J. H. Hormonal Regulation in Insects: Facts, Gaps, and Future Directions. Physiol. Rev. 1997, 77, 963– 1032,  DOI: 10.1152/physrev.1997.77.4.963
      There is no corresponding record for this reference.
    17. 17
      Willey, R. B. The Morphology of the Stomodeal Nervous System in Periplaneta americana (L.) and Other Blattaria. J. Morphol. 1961, 108, 219– 261,  DOI: 10.1002/jmor.1051080207
      17
      The morphology of the stomodeal nervous system in Periplaneta americana (L.) and other blattaria
      WILLEY R B
      Journal of morphology (1961), 108 (), 219-61 ISSN:0362-2525.
      There is no expanded citation for this reference.
    18. 18
      Predel, R.; Rapus, J.; Eckert, M. Myoinhibitory Neuropeptides in the American Cockroach. Peptides 2001, 22, 199– 208,  DOI: 10.1016/S0196-9781(00)00383-1
      18
      Myoinhibitory neuropeptides in the American cockroach
      Predel, R.; Rapus, J.; Eckert, M.
      Peptides (New York) (2001), 22 (2), 199-208CODEN: PPTDD5; ISSN:0196-9781. (Elsevier Science Inc.)
      A large no. of myostimulatory neuropeptides from neurohemal organs of the American cockroach (Periplaneta americana) have been described since 1989. These peptides, isolated from the retrocerebral complex and abdominal perisympathetic organs, are thought to be released as hormones. To study the coordinated action of these neuropeptides in the regulation of visceral muscle activity, it might be necessary to include myoinhibitors as well, however, not a single myoinhibitory neuropeptide of the American cockroach has been described so far. To fill this gap, the authors describe the isolation of LMS (leucomyosuppressin) and Pea-MIP (myoinhibitory peptide) from neurohemal organs of the American cockroach. LMS was very effective in inhibiting phasic activity of all visceral muscles tested. It was found in the corpora cardiaca of different species of cockroaches, as well as in related insect groups, including mantids and termites. Pea-MIP which is strongly accumulated in the corpora cardiaca was not detected with a muscle bioassay system but when searching for tryptophane-contg. peptides using a diode-array detector. This peptide caused only a moderate inhibition in visceral muscle assays. The distribution of Pea-MIP in neurohemal organs and cells supplying these organs with Pea-MIP immunoreactive material, is described. Addnl. to LMS and Pea-MIP, a member of the allatostatin peptide family, known to exhibit inhibitory properties in other insects, was tested in visceral muscle assays. This allatostatin was highly effective in inhibiting spontaneous activity of the foregut, but not of other tested visceral muscles of the American cockroach.
    19. 19
      Clynen, E.; Schoofs, L. Peptidomic Survey of the Locust Neuroendocrine System. Insect Biochem. Mol. Biol. 2009, 39, 491– 507,  DOI: 10.1016/j.ibmb.2009.06.001
      19
      Peptidomic survey of the locust neuroendocrine system
      Clynen, Elke; Schoofs, Liliane
      Insect Biochemistry and Molecular Biology (2009), 39 (8), 491-507CODEN: IBMBES; ISSN:0965-1748. (Elsevier Ltd.)
      Neuropeptides are important controlling agents in animal physiol. To understand their role and the ways in which neuropeptides behave and interact with one another, information on their time and sites of expression is required. The authors here used a combination of MALDI-TOF and ESI-Q-TOF mass spectrometry to make an inventory of the peptidome of different parts (ganglia and nerves) of the central nervous system from the desert locust Schistocerca gregaria and the African migratory locust Locusta migratoria. This way, the authors analyzed the brain, subesophageal ganglion, retrocerebral complex, stomatogastric nervous system, thoracic ganglia, abdominal ganglia and abdominal neurohemal organs. The result is an overview of the distribution of sixteen neuropeptide families, i.e., pyrokinins, pyrokinin-like peptides, periviscerokinins, tachykinins, allatotropin, accessory gland myotropin, FLRFamide, (short) neuropeptide F, allatostatins, insulin-related peptide co-peptide, ion-transport peptide co-peptide, corazonin, sulfakinin, orcokinin, hypertrehalosemic hormone and adipokinetic hormones (joining peptides) throughout the locust neuroendocrine system.
    20. 20
      Siegmund, T.; Korge, G. Innervation of the Ring Gland of Drosophila melanogaster. J. Comp. Neurol. 2001, 431, 481– 491,  DOI: 10.1002/1096-9861(20010319)431:4<481::AID-CNE1084>3.0.CO;2-7
      20
      Innervation of the ring gland of Drosophila melanogaster
      Siegmund T; Korge G
      The Journal of comparative neurology (2001), 431 (4), 481-91 ISSN:0021-9967.
      In insects, peptidergic neurons of the central nervous system regulate the synthesis of the main developmental hormones. Neuropeptides involved in this neuroendocrine cascade have been identified in lepidopterans and dictyopterans. Since these organisms are not suitable for genetic research, we identified peptidergic brain neurons innervating the ring gland in Drosophila melanogaster. In larvae of Drosophila, ecdysteroids and juvenile hormones are produced by the ring gland, which is composed of the prothoracic gland, the corpus allatum, and the corpora cardiaca. Using the GAL4 enhancer trap system, we mapped those neurons of the central nervous system that innervate the ring gland. Eleven groups of neurosecretory neurons and their target tissues were identified. Five neurons of the lateral protocerebrum directly innervate the prothoracic gland or corpus allatum cells of the ring gland and are believed to regulate ecdysteroid and juvenile hormone titers. Axons of the circadian pacemaker neurons project onto dendritic fields of these five neurons. This connection might be the neuronal substrate of the circadian rhythms of molting and metamorphosis in Drosophila. Most of the neurons presented here have not been described before. The enhancer trap lines labeling them will be valuable tools for the analysis of neuronal as well as genetic regulation in insect development.
    21. 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 Tagma-Specific Peptide Expression and Differential Processing. J. Neurochem. 2006, 96, 1362– 1374,  DOI: 10.1111/j.1471-4159.2005.03634.x
      21
      Direct mass spectrometric peptide profiling and fragmentation of larval peptide hormone release sites in Drosophila melanogaster reveals tagma-specific peptide expression and differential processing
      Wegener, Christian; Reinl, Tobias; Jaensch, Lothar; Predel, Reinhard
      Journal of Neurochemistry (2006), 96 (5), 1362-1374CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)
      Regulatory peptides represent a diverse group of messenger mols. In insects, they are produced by endocrine cells as well as secretory neurons within the CNS. Many regulatory peptides are released as hormones into the hemolymph to regulate, for example, diuresis, heartbeat or ecdysis behavior. Hormonal release of neuropeptides takes place at specialized organs, so-called neurohemal organs. The authors have performed a mass spectrometric characterization of the peptide complement of the main neurohemal organs and endocrine cells of the Drosophila melanogaster larva to gain insight into the hormonal communication possibilities of the fruit fly. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and MALDI-TOF-TOF tandem mass spectrometry, the authors detected 23 different peptides of which five were unpredicted by previous genome screenings. The authors also found a hitherto unknown peptide product of the capa gene in the ring gland and transverse nerves, suggesting that it might be released as hormone. The authors' results show that the peptidome of the neurohemal organs is tagma-specific and does not change during metamorphosis. The authors also provide evidence for the first case of differential prohormone processing in Drosophila.
    22. 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. Neonatal Insulin Action Impairs Hypothalamic Neurocircuit Formation in Response to Maternal High-Fat Feeding. Cell 2014, 156, 495– 509,  DOI: 10.1016/j.cell.2014.01.008
      22
      Neonatal Insulin Action Impairs Hypothalamic Neurocircuit Formation in Response to Maternal High-Fat Feeding
      Vogt, Merly C.; Paeger, Lars; Hess, Simon; Steculorum, Sophie M.; Awazawa, Motoharu; Hampel, Brigitte; Neupert, Susanne; Nicholls, Hayley T.; Mauer, Jan; Hausen, A. Christine; Predel, Reinhard; Kloppenburg, Peter; Horvath, Tamas L.; Bruening, Jens C.
      Cell (Cambridge, MA, United States) (2014), 156 (3), 495-509CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Maternal metabolic homeostasis exerts long-term effects on the offspring's health outcomes. Maternal high-fat diet (HFD) feeding during lactation predisposes the offspring for obesity and impaired glucose homeostasis in mice, which is assocd. with an impairment of the hypothalamic melanocortin circuitry. Whereas the no. and neuropeptide expression of anorexigenic proopiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP) neurons, electrophysiol. properties of POMC neurons, and posttranslational processing of POMC remain unaffected in response to maternal HFD feeding during lactation, the formation of POMC and AgRP projections to hypothalamic target sites is severely impaired. Abrogating insulin action in POMC neurons of the offspring prevents altered POMC projections to the preautonomic paraventricular nucleus of the hypothalamus (PVH), pancreatic parasympathetic innervation, and impaired glucose-stimulated insulin secretion in response to maternal overnutrition. These expts. reveal a crit. timing, when altered maternal metab. disrupts metabolic homeostasis in the offspring via impairing neuronal projections, and show that abnormal insulin signaling contributes to this effect.
    23. 23
      Trede, D.; Schiffler, S.; Becker, M.; Wirtz, S.; Steinhorst, K.; Strehlow, J.; Aichler, M.; Kobarg, J. H.; Oetjen, J.; Dyatlov, A. Exploring Three-Dimensional Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry Data: Three-Dimensional Spatial Segmentation of Mouse Kidney. Anal. Chem. 2012, 84, 6079– 6087,  DOI: 10.1021/ac300673y
      23
      Exploring Three-Dimensional Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry Data: Three-Dimensional Spatial Segmentation of Mouse Kidney
      Trede, Dennis; Schiffler, Stefan; Becker, Michael; Wirtz, Stefan; Steinhorst, Klaus; Strehlow, Jan; Aichler, Michaela; Kobarg, Jan Hendrik; Oetjen, Janina; Dyatlov, Andrey; Heldmann, Stefan; Walch, Axel; Thiele, Herbert; Maass, Peter; Alexandrov, Theodore
      Analytical Chemistry (Washington, DC, United States) (2012), 84 (14), 6079-6087CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Three-dimensional (3D) imaging has a significant impact on many challenges of life sciences. Three-dimensional matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is an emerging label-free bioanal. technique capturing the spatial distribution of hundreds of mol. compds. in 3D by providing a MALDI mass spectrum for each spatial point of a 3D sample. Currently, 3D MALDI-IMS cannot tap its full potential due to the lack efficient computational methods for constructing, processing, and visualizing large and complex 3D MALDI-IMS data. We present a new pipeline of efficient computational methods, which enables anal. and interpretation of a 3D MALDI-IMS data set. Construction of a MALDI-IMS data set was done according to the state-of-the-art protocols and involved sample prepn., spectra acquisition, spectra preprocessing, and registration of serial sections. For anal. and interpretation of 3D MALDI-IMS data, we applied the spatial segmentation approach which is well-accepted in anal. of two-dimensional (2D) MALDI-IMS data. In line with 2D data anal., we used edge-preserving 3D image denoising prior to segmentation to reduce strong and chaotic spectrum-to-spectrum variation. For segmentation, we used an efficient clustering method, called bisecting k-means, which is optimized for hierarchical clustering of a large 3D MALDI-IMS data set. Using the proposed pipeline, we analyzed a central part of a mouse kidney using 33 serial sections of 3.5 μm thickness after the PAXgene tissue fixation and paraffin embedding. For each serial section, a 2D MALDI-IMS data set was acquired following the std. protocols with the high spatial resoln. of 50 μm. Altogether, 512 495 mass spectra were acquired that corresponds to approx. 50 gigabytes of data. After registration of serial sections into a 3D data set, our computational pipeline allowed us to reveal the 3D kidney anatomical structure based on mass spectrometry data only. Finally, automated anal. discovered mol. masses colocalized with major anatomical regions. In the same way, the proposed pipeline can be used for anal. and interpretation of any 3D MALDI-IMS data set in particular of pathol. cases.
    24. 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,  DOI: 10.1021/acs.jproteome.8b00155
      24
      Transcriptomic and Neuropeptidomic Analysis of the Stick Insect, Carausius morosus
      Liessem, Sander; Ragionieri, Lapo; Neupert, Susanne; Bueschges, Ansgar; Predel, Reinhard
      Journal of Proteome Research (2018), 17 (6), 2192-2204CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      One of the most thoroughly studied insect species, with respect to locomotion behavior, is the stick insect Carausius morosus. Although detailed information exists on premotor networks controlling walking, surprisingly little is known about neuropeptides, which are certainly involved in motor activity generation and modulation. So far, only few neuropeptides were identified from C. morosus or related stick insects. We performed a transcriptome anal. of the central nervous system to assemble and identify 65 neuropeptide and protein hormone precursors of C. morosus, including 5 novel putative neuropeptide precursors without clear homol. to known neuropeptide precursors of other insects (Carausius neuropeptide-like precursor 1, HanSolin, PK-like1, PK-like2, RFLamide). Using Q Exactive Orbitrap and MALDI-TOF mass spectrometry, 277 peptides including 153 likely bioactive mature neuropeptides were confirmed. Peptidomics yielded a complete coverage for many of the neuropeptide propeptides and confirmed a surprisingly high no. of heterozygous sequences. Few neuropeptide precursors commonly occurring in insects, including those of insect kinins and sulfakinins, could neither be found in the transcriptome data nor did peptidomics support their presence. The results of our study represent 1 of the most comprehensive peptidomic analyses on insects and provide the necessary input for subsequent expts. revealing neuropeptide function in greater detail.
    25. 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,  DOI: 10.1021/ac048329g
      25
      Direct Molecular Imaging of Lymnaea stagnalis Nervous Tissue at Subcellular Spatial Resolution by Mass Spectrometry
      Altelaar, A. F. Maarten; Van Minnen, Jan; Jimenez, Connie R.; Heeren, Ron M. A.; Piersma, Sander R.
      Analytical Chemistry (2005), 77 (3), 735-741CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      The imaging capabilities of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and MALDI-MS sample prepn. methods were combined. We used this method, named matrix-enhanced (ME) SIMS, for direct mol. imaging of nervous tissue at micrometer spatial resoln. Cryosections of the cerebral ganglia of the freshwater snail Lymnaea stagnalis were placed on indium-tin-oxide (ITO)-coated conductive glass slides and covered with a thin layer of 2,5-dihydroxybenzoic acid by electrospray deposition. High-resoln. mol. ion maps of cholesterol and the neuropeptide APGWamide were constructed. APGWamide was predominantly localized in the cluster of neurons that regulate male copulation behavior of Lymnaea. ME-SIMS imaging allows direct mol.-specific imaging from tissue sections without labeling and opens a complementary mass window (<2500 Da) to MALDI imaging mass spectrometry at an order of magnitude higher spatial resoln. (<3 μm).
    26. 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,  DOI: 10.1002/jms.505
      26
      Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: Practical aspects of sample preparation
      Schwartz, Sarah A.; Reyzer, Michelle L.; Caprioli, Richard M.
      Journal of Mass Spectrometry (2003), 38 (7), 699-708CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)
      Practical guidelines for the prepn. of tissue sections for direct anal. by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry are presented. Techniques for proper sample handling including tissue storage, sectioning and mounting are described. Emphasis is placed on optimizing matrix parameters such as the type of matrix mol. used, matrix concn., and solvent compn. Several different techniques for matrix application are illustrated. Optimal instrument parameters and the necessity for advanced data anal. approaches with regards to direct tissue anal. are also discussed.
    27. 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,  DOI: 10.1016/j.jasms.2008.03.016
      27
      Enhancement of Protein Sensitivity for MALDI Imaging Mass Spectrometry After Chemical Treatment of Tissue Sections
      Seeley, Erin H.; Oppenheimer, Stacey R.; Mi, Deming; Chaurand, Pierre; Caprioli, Richard M.
      Journal of the American Society for Mass Spectrometry (2008), 19 (8), 1069-1077CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
      MALDI imaging mass spectrometry (IMS) has become a valuable tool for the investigation of the content and distribution of mol. species in tissue specimens. Numerous methodol. improvements have been made to optimize tissue section prepn. and matrix deposition protocols, as well as MS data acquisition and processing. In particular for proteomic analyses, washing the tissue sections before matrix deposition has proven useful to improve spectral qualities by increasing ion yields and the no. of signals obsd. The authors systematically explore here the effects of several solvent combinations for washing tissue sections. To minimize exptl. variability, all of the measurements were performed on serial sections cut from a single mouse liver tissue block. Several other key steps of the process such as matrix deposition and MS data acquisition and processing have also been automated or standardized. To assess efficacy, after each washing procedure the total ion current and no. of peaks were counted from the resulting protein profiles. These results were correlated to on-tissue measurements obtained for lipids. Using similar approaches, several selected washing procedures were also tested for their ability to extend the lifetime as well as revive previously cut tissue sections. The effects of these washes on automated matrix deposition and crystn. behavior as well as their ability to preserve tissue histol. were also studied. Finally, in a full-scale IMS study, these washing procedures were tested on a human renal cell carcinoma biopsy.
    28. 28
      Meding, S.; Walch, A. MALDI Imaging Mass Spectrometry for Direct Tissue Analysis. Methods Mol. Biol. 2012, 931, 537– 546,  DOI: 10.1007/978-1-62703-056-4_29
      There is no corresponding record for this reference.
    29. 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,  DOI: 10.1021/acs.jproteome.8b00098
      29
      MALDI Imaging Analysis of Neuropeptides in Africanized Honeybee (Apis mellifera) Brain: Effect of Aggressiveness
      Pratavieira, Marcel; Menegasso, Anally Ribeiro da Silva; Esteves, Franciele Grego; Sato, Kenny Umino; Malaspina, Osmar; Palma, Mario Sergio
      Journal of Proteome Research (2018), 17 (7), 2358-2369CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Aggressiveness in honeybees seems to be regulated by multiple genes, under the influence of different factors, such as polyethism of workers, environmental factors, and response to alarm pheromones, creating a series of behavioral responses. It is suspected that neuropeptides seem to be involved with the regulation of the aggressive behavior. The role of allatostatin and tachykinin-related neuropeptides in honeybee brain during the aggressive behavior is unknown, and thus worker honeybees were stimulated to attack and to sting leather targets hung in front of the colonies. The aggressive individuals were collected and immediately frozen in liq. nitrogen; the heads were removed and sliced at sagittal plan. The brain slices were submitted to MALDI spectral imaging anal., and the results of the present study reported the processing of the precursors proteins into mature forms of the neuropeptides AmAST A (59-76) (AYTYVSEYKRLPVYNFGL-NH2), AmAST A (69-76) (LPVYNFGL-NH2), AmTRP (88-96) (APMGFQGMR-NH2), and AmTRP (254-262) (ARMGFHGMR-NH2), which apparently acted in different neuropils of the honeybee brain during the aggressive behavior, possibly taking part in the neuromodulation of different aspects of this complex behavior. These results were biol. validated by performing aggressiveness-related behavioral assays using young honeybee workers that received 1 ng of AmAST A (69-76) or AmTRP (88-96) via hemocele. The young workers that were not expected to be aggressive individuals presented a complete series of aggressive behaviors in the presence of the neuropeptides, corroborating the hypothesis that correlates the presence of mature AmASTs A and AmTRPs in the honeybee brain with the aggressiveness of this insect.
    30. 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,  DOI: 10.1021/ac9507956
      30
      Influence of Matrix Solution Conditions on the MALDI-MS Analysis of Peptides and Proteins
      Cohen, Steven L.; Chait, Brian T.
      Analytical Chemistry (1996), 68 (1), 31-7CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Sample-matrix prepn. procedures are shown to greatly influence the quality of the matrix-assisted laser desorption/ionization (MALDI) mass spectra of peptides and proteins. In particular, dramatic mass discrimination effects are obsd. when the matrix 4-hydroxy-α-cyanocinnamic acid is used for analyzing complex mixts. of peptides and proteins. The discrimination effects are strongly dependent on the sample-matrix soln. compn., pH, and the rates at which the sample-matrix cocrystals are grown. These findings demonstrate the need to exercise great care in performing and interpreting the MALDI anal. of biol. samples. The results also indicate that there is a reverse-phase chromatog.-like dimension in the sample-matrix prepn. procedures that can be exploited to optimize the anal. The present work describes the conditions under which the majority of components of a complex mixt. of peptides and proteins can be successfully measured.
    31. 31
      Ong, T.-H.; Romanova, E. V.; Roberts-Galbraith, R. H.; Yang, N.; 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,  DOI: 10.1074/jbc.M115.709196
      There is no corresponding record for this reference.
    32. 32
      Sadeghi, M.; Vertes, A. Crystallite Size Dependence of Volatilization in Matrix-Assisted Laser Desorption Ionization. Appl. Surf. Sci. 1998, 127–129, 226– 234,  DOI: 10.1016/S0169-4332(97)00636-3
      32
      Crystallite size dependence of volatilization in matrix-assisted laser desorption ionization
      Sadeghi, Mehrnoosh; Vertes, Akos
      Applied Surface Science (1998), 127-129 (), 226-234CODEN: ASUSEE; ISSN:0169-4332. (Elsevier Science B.V.)
      During the desorption and ionization processes in matrix-assisted laser desorption ionization (MALDI), the laser beam interacts with different areas of the polycryst. sample surface varying in crystal size and structure. CCD imaging of dried-droplet and electrospray deposited MALDI samples was used to explore the effect of laser exposure on surface morphol. at atm. pressure. Crystal size distributions of the sinapinic acid target were examd. prior to and after laser irradn. Image processing and anal. of the difference images revealed the morphol. changes in the sample. Near-threshold irradiance smaller crystals (∼2 μm) are completely volatilized by the laser shot, whereas larger crystals undergo layer-by-layer evapn. (peeling). Heat conduction simulations in a finite slab demonstrated that under similar conditions, the surface temp. of small crystallites increases markedly compared to their larger counterparts. These higher surface temps. can lead to the selective volatilization of smaller crystallites. This study points to the influence of sample prepn. on the crystal size distribution and its consequence on volatilization in MALDI-mass spectrometry (MS).
    33. 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,  DOI: 10.1007/s00216-014-8305-7
      33
      Towards imaging metabolic pathways in tissues
      Dekker, Tim J. A.; Jones, Emrys A.; Corver, Willem E.; van Zeijl, Rene J. M.; Deelder, Andre M.; Tollenaar, Rob A. E. M.; Mesker, Wilma E.; Morreau, Hans; McDonnell, Liam A.
      Analytical and Bioanalytical Chemistry (2015), 407 (8), 2167-2176CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging using 9-aminoacridine as the matrix leads to the detection of low mass metabolites and lipids directly from cancer tissues. These included lactate and pyruvate for studying the Warburg effect, as well as succinate and fumarate, metabolites whose accumulation is assocd. with specific syndromes. By using the pathway information present in the human metabolome database, it was possible to identify regions within tumor tissue samples with distinct metabolic signatures that were consistent with known tumor biol. The authors present a data anal. workflow for assessing metabolic pathways in their histopathol. context.
    34. 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. Multimodal Mass Spectrometry Imaging of N-Glycans and Proteins from the Same Tissue Section. Anal. Chem. 2016, 88, 7745– 7753,  DOI: 10.1021/acs.analchem.6b01739
      34
      Multimodal Mass Spectrometry Imaging of N-Glycans and Proteins from the Same Tissue Section
      Heijs, Bram; Holst, Stephanie; Briaire-de Bruijn, Inge H.; van Pelt, Gabi W.; de Ru, Arnoud H.; van Veelen, Peter A.; Drake, Richard R.; Mehta, Anand S.; Mesker, Wilma E.; Tollenaar, Rob A.; Bovee, Judith V. M. G.; Wuhrer, Manfred; McDonnell, Liam A.
      Analytical Chemistry (Washington, DC, United States) (2016), 88 (15), 7745-7753CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated mol. information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the in situ multimodal anal. of N-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major anal. aspects, such as lateral diffusion of the analyte mols. and differences in measurement sensitivity due to the addnl. sample prepn. methods, have been investigated for both N-glycans and proteolytic peptides. By combining the MSI approach with ext. anal., we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique N-glycan and peptide identities.
    35. 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, 208, 283– 297,  DOI: 10.1007/BF00234877
      35
      Identification of neuronal pathways between the stomatogastric nervous system and the retrocerebral complex of the cockroach Periplaneta americana (L.)
      Gundel M; Penzlin H
      Cell and tissue research (1980), 208 (2), 283-97 ISSN:0302-766X.
      The neuronal pathways connecting the stomatogastric nervous system with the retrocerebral complex of the cockroach, Periplaneta americana, were investigated by means of axonal cobalt chloride iontophoresis. Somata in the hypocerebral ganglion and in the nervus recurrens sending their axons to different parts of the stomatogastric nervous system were traced. Some axons in the oesophageal nerve arise from large perikarya in the anterior part of the pars intercerebralis and pass via the NCCI to the corpora cardiaca and the oesophageal nerve. The form a profuse dendritic tree in the protocerebrum. Fibers of the NCC I and NCC II as well as the NCA I and NCA II enter the stomatogastric nervous system via the hypocerebral ganglion.
    36. 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,  DOI: 10.1016/0020-7322(84)90033-3
      There is no corresponding record for this reference.
    37. 37
      Predel, R. Peptidergic Neurohemal System of an Insect: Mass Spectrometric Morphology. J. Comp. Neurol. 2001, 436, 363– 375,  DOI: 10.1002/cne.1073
      37
      Peptidergic neurohemal system of an insect: Mass spectrometric morphology
      Predel, Reinhard
      Journal of Comparative Neurology (2001), 436 (3), 363-375CODEN: JCNEAM; ISSN:0021-9967. (Wiley-Liss, Inc.)
      Neuropeptides are by far the most diverse group of messenger mols. in insects. To understand cell signaling and function, it is essential to reveal the complete neuropeptide profile of a single neuron/nerve/neurohemal organ first. In this study, matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry was used to analyze the peptidergic system of an insect, focusing on the neurohemal structures. Major neurohemal organs were investigated, including the retrocerebral complex, perisympathetic organs, and all nerves supplying these organs with neurosecretions. Addnl., peripheral neurohemal release sites such as the dilator muscle of the antennal circulatory organ and lateral heart nerves were studied, as well as parts of the stomatogastric nervous system. The following neuropeptide families were analyzed: kinins, allatostatins, leucomyosuppressin, corazonin, adipokinetic hormones, myoinhibitory peptide, sulfakinins, periviscerokinins, YLSamide, VEAacid, SKNacid, proctolin, the head peptide, and pyrokinins. Beyond a contribution to a map of the distribution of neuropeptides in a neurohemal system, the following conclusions can be drawn from these expts. (1) Nearly all abundant peaks in the different mass spectra represent peptides that have already been identified. (2) Although only adult males were used in this study, variations in the peptide abundances were obsd. that are possibly correlated with different physiol./developmental conditions. (3) Peptides have a body-region-specific distribution in the neurohemal system. (4) A clear compartmentalization of the retrocerebral complex could be obsd.
    38. 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,  DOI: 10.1002/cne.903220105
      38
      Neurons in the cockroach nervous system reacting with antisera to the neuropeptide leucokinin I
      Nassel D R; Cantera R; Karlsson A
      The Journal of comparative neurology (1992), 322 (1), 45-67 ISSN:0021-9967.
      Antisera were raised against the myotropic neuropeptide leucokinin I, originally isolated from head extracts of the cockroach Leucophaea maderae. Processes of leucokinin I immunoreactive (LKIR) neurons were distributed throughout the nervous system, but immunoreactive cell bodies were not found in all neuromeres. In the brain, about 160 LKIR cell bodies were distributed in the protocerebrum and optic lobes (no LKIR cell bodies were found in the deuto- and tritocerebrum). In the ventral ganglia, LKIR cell bodies were seen distributed as follows: eight (weakly immunoreactive) in the subesophageal ganglion; about six larger and bilateral clusters of 5 smaller in each of the three thoracic ganglia, and in each of the abdominal ganglia, two pairs of strongly immunoreactive cell bodies were resolved. Many of the LKIR neurons could be described in detail. In the optic lobes, immunoreactive neurons innervate the medulla and accessory medulla. In the brain, three pairs of bilateral LKIR neurons supply branches to distinct sets of nonglomerular neuropil, and two pairs of descending neurons connect the posterior protocerebrum to the antennal lobes and all the ventral ganglia. Other brain neurons innervate the central body, tritocerebrum, and nonglomerular neuropil in protocerebrum. LKIR neurons of the median and lateral neurosecretory cell groups send axons to the corpora cardiaca, frontal ganglion, and tritocerebrum. In the muscle layer of the foregut (crop), bi- and multipolar LKIR neurons with axons running to the retrocerebral complex were resolved. The LKIR neurons in the abdominal ganglia form efferent axons supplying the lateral cardiac nerves, spiracles, and the segmental perivisceral organs. The distribution of immunoreactivity indicates roles for leucokinins as neuromodulators or neurotransmitters in central interneurons arborizing in different portions of the brain, visual system, and ventral ganglia. Also, a function in circuits regulating feeding can be presumed. Furthermore, a role in regulation of heart and possibly respiration can be suggested, and probably leucokinins are released from corpora cardiaca as neurohormones. Leucokinins were isolated by their myotropic action on the Leucophaea hindgut, but no innervation of this portion of the gut could be demonstrated. The distribution of leucokinin immunoreactivity was compared to immunolabeling with antisera against vertebrate tachykinins and lysine vasopressin.
    39. 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,  DOI: 10.1021/acs.jproteome.7b00630
      39
      Neuropeptidomics of the Bed Bug Cimex lectularius
      Predel, Reinhard; Neupert, Susanne; Derst, Christian; Reinhardt, Klaus; Wegener, Christian
      Journal of Proteome Research (2018), 17 (1), 440-454CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      The bed bug Cimex lectularius is a globally distributed human ectoparasite with fascinating biol. It has recently acquired resistance against a broad range of insecticides, causing a worldwide increase in bed bug infestations. The recent annotation of the bed bug genome revealed a full complement of neuropeptide and neuropeptide receptor genes in this species. With regard to the biol. of C. lectularius, neuropeptide signaling is esp. interesting because it regulates feeding, diuresis, digestion, as well as reprodn. and also provides potential new targets for chem. control. To identify which neuropeptides are translated from the genome-predicted genes, we performed a comprehensive peptidomic anal. of the central nervous system of the bed bug. We identified in total 144 different peptides from 29 precursors, of which at least 67 likely present bioactive mature neuropeptides. C. lectularius corazonin and myosuppressin are unique and deviate considerably from the canonical insect consensus sequences. Several identified neuropeptides likely act as hormones, as evidenced by the occurrence of resp. mass signals and immunoreactivity in neurohemal structures. Our data provide the most comprehensive peptidome of a Heteropteran species so far and in comparison suggest that a hematophageous life style does not require qual. adaptations of the insect peptidome.
    40. 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,  DOI: 10.1021/pr5011636
      40
      Neuropeptidomics of the Carpenter Ant Camponotus floridanus
      Schmitt, Franziska; Vanselow, Jens T.; Schlosser, Andreas; Kahnt, Joerg; Roessler, Wolfgang; Wegener, Christian
      Journal of Proteome Research (2015), 14 (3), 1504-1514CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Ants show a rich behavioral repertoire and a highly complex organization, which have been attracting behavioral and sociobiol. researchers for a long time. The neuronal underpinnings of ant behavior and social organization are, however, much less understood. Neuropeptides are key signals that orchestrate animal behavior and physiol., and it is thus feasible to assume that they play an important role also for the social constitution of ants. Despite the availability of different ant genomes and in silico prediction of ant neuropeptides, a comprehensive biochem. survey of the neuropeptidergic communication possibilities of ants is missing. We therefore combined different mass spectrometric methods to characterize the neuropeptidome of the adult carpenter ant Camponotus floridanus. We also characterized the local neuropeptide complement in different parts of the nervous and neuroendocrine system, including the antennal and optic lobes. Our anal. identifies 39 neuropeptides encoded by different prepropeptide genes, and in silico predicts new prepropeptide genes encoding CAPA peptides, CNMamide as well as homologs of the honey bee IDLSRFYGHFNT- and ITGQGNRIF-contg. peptides. Our data provides basic information about the identity and localization of neuropeptides that is required to anatomically and functionally address the role and significance of neuropeptides in ant behavior and physiol.
    41. 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. 2004, 88, 311– 319,  DOI: 10.1046/j.1471-4159.2003.02161.x
      41
      Expression of a novel neuropeptide, NVGTLARDFQLPIPNamide, in the larval and adult brain of Drosophila melanogaster
      Verleyen, Peter; Baggerman, Geert; Wiehart, Ursula; Schoeters, Eric; Van Lommel, Alfons; De Loof, Arnold; Schoofs, Liliane
      Journal of Neurochemistry (2004), 88 (2), 311-319CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)
      Advances in mass spectrometry and the availability of genomic databases made it possible to det. the peptidome or peptide content of a specific tissue. Peptidomics by nanoflow capillary liq. chromatog. tandem mass spectrometry of an ext. of 50 larval Drosophila brains, yielded 28 neuropeptides. Eight were entirely novel and encoded by five not yet annotated genes; only two genes had a homolog in the Anopheles gambiae genome. Seven of the eight peptides did not show relevant sequence homol. to any known peptide. Therefore, no evidence towards the physiol. role of these "orphan" peptides was available. We identified one of the eight peptides, IPNamide, in an ext. of the Drosophila adult brain as well. Next, specific antisera were raised to reveal the distribution pattern of IPNamide and other peptides from the same precursor, in larval and adult brains by means of whole-mount immunocytochem. and confocal microscopy. IPNamide immunoreactivity is abundantly present in both stages and a striking similarity was found between the distribution patterns of IPNamide and TPAEDFMRFamide, a member of the FMRFamide peptide family. Based on this distribution pattern, IPNamide might be involved in phototransduction, in processing sensory stimuli, as well as in controlling the activity of the esophagus.
    42. 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-Enkephalin-Arg6-Gly7-Leu8. Cell Tissue Res. 1991, 263, 285– 291,  DOI: 10.1007/BF00318770
      42
      Immunocytochemical mapping of neuronal pathways from brain to corpora cardiaca/corpora allata in the cockroach Diploptera punctata with antisera against Met-enkephalin-Arg6-Gly7-Leu8
      Duve H; Thorpe A; Tobe S S
      Cell and tissue research (1991), 263 (2), 285-91 ISSN:0302-766X.
      Neuronal circuits in the brain and retrocerebral complex of the cockroach Diploptera punctata have been mapped immunocytochemically with antisera directed against the extended enkephalin, Met-enkephalin-Arg6-Gly7-Leu8 (Met-8). The pathways link median and lateral neurosecretory cells with the corpus cardiacum corpus allatum complex. In females, nerve fibres penetrate the corpora allata and varicosities or terminals, immunoreactive to Met-8, surround the glandular cells. Males differ in having almost no Met-8 immunoreactivity in the corpora allata. The corpora cardiaca of both males and females are richly supplied with Met-8 immunoreactive material, in particular in the 'cap' regions immediately adjacent to the corpora allata. A similarity in the amino-acid sequences of Met-8 and the C-terminus of the recently characterised allatostatins of D. punctata suggests that the pathways identified with the Met-8 antisera may be the same as those by which the allatostatins are transported from the brain to the corpus allatum. In comparative studies on the blowfly Calliphora vomitoria, similar neuronal pathways have been identified except that no sexual dimorphism with respect to amounts of immunoreactive material within the corpus allatum has been observed. These results suggest a possible homology in the neuropeptide regulation of the gland.
    43. 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,  DOI: 10.1007/BF00327985
      43
      Localization of corazonin in the nervous system of the cockroach Periplaneta americana
      Veenstra, Jan A.; Davis, Norman T.
      Cell & Tissue Research (1993), 274 (1), 57-64CODEN: CTSRCS; ISSN:0302-766X.
      Antisera raised to the cardioactive peptide corazonin were used to localize immunoreactive cells in the nervous system of the American cockroach. Sera obtained after the seventh booster injection were sufficiently specific to be used for immunocytol. They recognized a subset of 10 lateral neurosecretory cells in the proto-cerebrum that project to, and arborize and terminate in the ipsilateral corpus cardiacum. They also reacted with bilateral neurons in each of the thoracic and abdominal neuromeres, a single dorsal unpaired median neuron in the subesophageal ganglion, an interneuron in each optic lobe, and other neurons at the base of the optic lobe, in the tritocerebrum and deutocerebrum. The presence of corazonin in the abdominal neurons and the lateral neurosecretory cells was confirmed by HPLC fractionation of exts. of the abdominal ganglia, brains and retrocerebral complexes, followed by detn. of corazonin by ELISA, which revealed in each tissue a single immunoreactive peak co-eluting with corazonin in two different HPLC systems. Antisera obtained after the first three booster injections recognized a large no. of neuroendocrine cells and neurons in the brain and the abdominal nerve cord. However, the sera from the two rabbits reacted largely with different cells, indicating that the majority of this immunoreactivity was due to cross-reactivity. These results indicate that the prodn. of highly specific antisera to some neuropeptides may require a considerable no. of booster injections.
    44. 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,  DOI: 10.1002/cne.21183
      44
      Peptidomics of identified neurons demonstrates a highly differentiated expression pattern of FXPRLamides in the neuroendocrine system of an insect
      Predel Reinhard; Eckert Manfred; Pollak Edit; Molnar Laszlo; Scheibner Olaf; Neupert Susanne
      The Journal of comparative neurology (2007), 500 (3), 498-512 ISSN:0021-9967.
      FXPRLamides are insect neuropeptides that mediate such diverse functions as pheromone biosynthesis, visceral muscle contraction, and induction of diapause. Although multiple forms occur in every insect studied so far, little is known about a possible functional differentiation and/or differences in the cellular expression pattern of these messenger molecules. In this study, we performed a mass spectrometric survey of all FXPRLamide-expressing neurosecretory neurons in the CNS of Periplaneta americana. That species combines a very well characterized peptidergic system with relatively easy accessible neurosecretory cells suitable for dissection. In addition to the extensive mass spectrometric analyses of single cells, the projection of the FXPRLamide-expressing neurons was studied with three antisera specifically recognizing different FXPRLamides. The following conclusions can be drawn from this first comprehensive peptidomic approach on insect neurons. 1) A high degree of differentiation in the expression of FXPRLamides exists; not fewer then four cell types containing different sets of FXPRLamides were observed. 2) A low level of colocalization with other neuropeptides was found in these neurons. 3) A comparison with FXPRLamide-expressing neurons of other insects shows a high degree of conservation in the localization and projection of these neurons, which is not corroborated by a similar conservation of the corresponding peptide sequences. 4) Although the methods for cell identification, dissection, and sample preparation for mass spectrometry were kept as simple as possible, it was unambiguously shown that this approach is generally suitable for routine analysis of single identified neurons of insects.
    45. 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,  DOI: 10.1016/j.jasms.2007.06.010
      45
      Sublimation as a Method of Matrix Application for Mass Spectrometric Imaging
      Hankin, Joseph A.; Barkley, Robert M.; Murphy, Robert C.
      Journal of the American Society for Mass Spectrometry (2007), 18 (9), 1646-1652CODEN: JAMSEF; ISSN:1044-0305. (Elsevier Inc.)
      Common org. matrix-assisted laser desorption/ionization (MALDI) matrixes, 2,5-dihydroxybenzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, and α-cyano-4-hydroxycinnamic acid, were found to undergo sublimation without decompn. under conditions of reduced pressure and elevated temp. This solid to vapor-phase transition was exploited to apply MALDI matrix onto tissue samples over a broad surface in a solvent-free application for mass spectrometric imaging. Sublimation of matrix produced an even layer of small crystals across the sample plate. The deposition was readily controlled with time, temp., and pressure settings and was highly reproducible from one sample to the next. Mass spectrometric images acquired from phospholipid stds. robotically spotted onto a MALDI plate yielded a more intense, even signal with fewer sodium adducts when matrix was applied by sublimation relative to samples where matrix was deposited by an electrospray technique. MALDI matrix could be readily applied to tissue sections on glass slides and stainless steel MALDI plate inserts as long as good thermal contact was made with the condenser of the sublimation device. Sections of mouse brain were coated with matrix applied by sublimation and were imaged using a Q-q-TOF mass spectrometer to yield mass spectral images of very high quality. Image quality is likely enhanced by several features of this technique including the microcryst. morphol. of the deposited matrix, increased purity of deposited matrix, and evenness of deposition. This inexpensive method was reproducible and eliminated the potential for spreading of analytes arising from solvent deposition during matrix application.
    46. 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,  DOI: 10.1021/ac200998a
      46
      Matrix Sublimation/Recrystallization for Imaging Proteins by Mass Spectrometry at High Spatial Resolution
      Yang, Junhai; Caprioli, Richard M.
      Analytical Chemistry (Washington, DC, United States) (2011), 83 (14), 5728-5734CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      We have employed matrix deposition by sublimation for protein image anal. on tissue sections using a hydration/recrystn. process that produces high-quality MALDI mass spectra and high-spatial-resoln. ion images. We systematically investigated different washing protocols, the effect of tissue section thickness, the amt. of sublimated matrix per unit area, and different recrystn. conditions. The results show that an org. solvent rinse followed by ethanol/water rinses substantially increased sensitivity for the detection of proteins. Both the thickness of the tissue section and the amt. of sinapinic acid sublimated per unit area have optimal ranges for maximal protein signal intensity. Ion images of mouse and rat brain sections at 50, 20, and 10 μm spatial resoln. are presented and are correlated with hematoxylin and eosin (H&E)-stained optical images. For targeted anal., histol.-directed imaging can be performed using this protocol where MS anal. and H&E staining are performed on the same section.
    47. 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,  DOI: 10.1038/nmeth.4433
      47
      Autofocusing MALDI mass spectrometry imaging of tissue sections and 3D chemical topography of nonflat surfaces
      Kompauer, Mario; Heiles, Sven; Spengler, Bernhard
      Nature Methods (2017), 14 (12), 1156-1158CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      We describe an atm. pressure matrix-assisted laser desorption-ionization mass spectrometry imaging system that uses long-distance laser triangulation on a micrometer scale to simultaneously obtain topog. and mol. information from 3D surfaces. We studied the topog. distribution of compds. on irregular 3D surfaces of plants and parasites, and we imaged nonplanar tissue sections with high lateral resoln., thereby eliminating height-related signal artifacts.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b04304.

    • List of mature neuropeptides from 15 precursor genes of P. americana; quadrupole orbitrap MS2 spectra of P. americana neuropeptides; workflow for MSI sample preparation optimized for insect neuroendocrine tissue (RCC); comparison of peptide coverage in tissue sections dried for 1 or 12 h before washing; comparison of peptide coverage in tissue sections with and without successive ethanol washes; comparison of peptide coverage in tissue sections after matrix spraying (5 mg/mL CHCA in 50% ACN/H2O) with matrix solution containing 0.1 or 2% TFA; distribution of allatostatinA-11, corazonin, and allatotropin in consecutive RCC sections; MALDI-TOF direct tissue profiling of a dissected nervus corporis cardiaci 1; and discrimination between mass-similar neuropeptides (PDF)


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