Name: multi-faceted mass spectrometric investigation of neuropeptides in callinectes sapidus
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This research was supported by National Science Foundation (CHE-1710140 and CHE-2108223) and National Institutes of Health (NIH) through grant R01DK071801. A.P. was supported in part by the NIH Chemistry-Biology Interface Training Grant (T32 GM008505). N.V.Q. was supported in part by the National Institutes of Health, under the Ruth L. Kirschstein National Research Service Award from the National Heart Lung and Blood Institute to the University of Wisconsin-Madison Cardiovascular Research Center (T32 HL007936). L.L. would like to acknowledge NIH grants R56 MH110215, S10RR029531, and S10OD025084, as well as funding support from a Vilas Distinguished Achievement Professorship and Charles Melbourne Johnson Professorship with funding provided by the Wisconsin Alumni Research Foundation and University of Wisconsin-Madison School of Pharmacy.

All tissue sampling described was performed in compliance with the University of Wisconsin-Madison guidelines.
1. LC-ESI-MS analysis of neuropeptides
<ol>
	<li>Neuropeptide extraction and desalting
	<ol>
		<li>Prior to tissue acquisition, prepare acidified methanol (acMeOH) (90:9:1 MeOH:water:acetic acid) as described in16.</li>
		<li>Collect brain tissue from the crustacean17 and use forceps to immediately place one tissue eac...

<ol>
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A. 1360, 217-228 (2014).</li><li>Li, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanosecond+photochemically+promoted+click+chemistry+for+enhanced+neuropeptide+visualization+and+rapid+protein+labeling.">Nanosecond photochemically promoted click chemistry for enhanced neuropeptide visualization and rapid protein labeling.</a> Nature Communications. 10 (1), 4697 (2019).</li><li>Corbi&#232;re, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+the+Identification+of+Bioactive+Neuropeptides+in+Vertebrates.">Strategies for the Identification of Bioactive Neuropeptides in Vertebrates.</a> Frontiers in Neuroscience. 13, 948 (2019).</li><li>Chen, R., Ma, M., Hui, L., Zhang, J., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+neuropeptides+in+crustacean+hemolymph+via+MALDI+mass+spectrometry.">Measurement of neuropeptides in crustacean hemolymph via MALDI mass spectrometry.</a> Journal of American Society for Mass Spectrometry. 20 (4), 708-718 (2009).</li><li>Fridjonsdottir, E., Nilsson, A., Wadensten, H., Andr&#233;n, P. 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The accurate identification, quantification, and localization of neuropeptides and endogenous peptides found in the nervous system are crucial toward understanding their function23,24. Mass spectrometry is a powerful technique that can allow all of this to be accomplished, even in organisms without a fully sequenced genome. The ability of this protocol to detect, quantify, and localize neuropeptides from tissue collected from C. sapidus through a combina...

The nervous system is complex and requires a network of neurons to transmit signals throughout an organism. The nervous system coordinates sensory information and biological response. The intricate and convoluted interactions involved in signal transmission require many different signaling molecules such as neurotransmitters, steroids, and neuropeptides. As neuropeptides are the most diverse and potent signaling molecules that play key roles in activating physiological responses to stress and other stimuli, it is of interest to determine their specific role in these physiological processes. Neuropeptide function is related to their amino acid structure, which determines mobility, receptor interaction, and affinity1. Techniques such as histochemistry, which is important because neuropeptides can be synthesized, stored, and released in different regions of the tissue, and electrophysiology have been employed to investigate neuropeptide structure and function2,3,4, but these methods are limited by throughput and specificity to resolve the vast sequence diversity of neuropeptides.
Mass spectrometry (MS) enables the high throughput analysis of neuropeptide structure and abundance. This can be performed through different MS techniques, most commonly liquid chromatography-electrospray ionization MS (LC-ESI-MS)5 and matrix-assisted laser desorption/ionization MS (MALDI-MS)6. Utilizing high accuracy mass measurements and MS fragmentation, MS provides the ability to assign amino acid sequence and post-translational modification (PTM) status to neuropeptides from complex mixtures without a priori knowledge to aid in ascertaining their function7,8. In addition to qualitative information, MS enables quantitative information of neuropeptides through label-free quantitation (LFQ) or label-based methods such as isotopic or isobaric labeling9. The main advantages of LFQ include its simplicity, low cost of analysis, and decreased sample preparation steps which can minimize sample loss. However, the disadvantages of LFQ include increased instrument time costs as it requires multiple technical replicates to address quantitative error from run-to-run variability. This also leads to a decreased ability to accurately quantify small variations. Label-based methods are subjected to less systematic variation as multiple samples can be differentially labeled using a variety of stable isotopes, combined into one sample, and analyzed through mass spectrometry simultaneously. This also increases throughput, although isotopic labels can be time consuming and costly to synthesize or purchase. Full scan mass spectra (MS1) spectral complexity also increases as multiplexing increases, which decreases the number of unique neuropeptides able to be fragmented and therefore, identified. Conversely, isobaric labeling does not increase spectral complexity at the MS1 level, although it introduces challenges for low abundance analytes such as neuropeptides. As isobaric quantitation is performed at the fragment ion mass spectra (MS2) level, low-abundance neuropeptides may be unable to be quantified as more abundant matrix components may be selected for fragmentation and those selected may not have high enough abundance to be quantified. With isotopic labeling, quantitation can be performed on every identified peptide.
In addition to identification and quantification, localization information can be obtained by MS through MALDI-MS imaging (MALDI-MSI)10. By rastering a laser across a sample surface, MS spectra can be compiled into a heat map image for each m/z value. Mapping transient neuropeptide signal intensity in different regions across conditions can provide valuable information for function determination11. Localization of neuropeptides is especially important because neuropeptide function may differ depending on location12.
Neuropeptides are found in lower abundance in vivo than other signaling molecules, such as neurotransmitters, and thus require sensitive methods for detection13. This can be achieved through the removal of higher abundance matrix components, such as lipids11,14. Additional considerations for the analysis of neuropeptides need to be made when compared to common proteomics workflows, mainly because most neuropeptidomic analyses omit enzymatic digestion. This limits software options for neuropeptide data analysis as most were built with algorithms based on proteomics data and protein matches informed by peptide detection. However, many software such as PEAKS15 is more suited to neuropeptide analysis due to their de novo sequencing capabilities. Several factors need to be considered for the analysis of neuropeptides starting from extraction method to MS data analysis.
The protocols described here include methods for sample preparation and dimethyl isotopic labeling, data acquisition, and data analysis of neuropeptides by LC-ESI-MS, MALDI-MS, and MALDI-MSI. Through representative results from several experiments, the utility and ability of these methods to identify, quantify, and localize neuropeptides from blue crabs, Callinectes sapidus, is demonstrated. To better understand the nervous system, model systems are commonly used. Many organisms do not have a fully sequenced genome available, which prevents comprehensive neuropeptide discovery at the peptide level. In order to mitigate this challenge, a protocol for identifying novel neuropeptides and transcriptome mining to generate databases for organisms without complete genome information is included. All protocols presented here can be optimized for neuropeptide samples from any species, as well as applied for the analysis of any endogenous peptides.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals, Reagents, and Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2,5-Dihydroxybenzoic acid (DHB) matrix</td>
			<td>Supelco</td>
			<td>39319</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Fisher Chemical</td>
			<td>A38S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile Optima LC/MS grade</td>
			<td>Fisher Chemical</td>
			<td>A955-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>Borane pyridine</td>
			<td>Sigma-Aldrich</td>
			<td>179752</td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker peptide calibration mix</td>
			<td>Bruker Daltonics</td>
			<td>NC9846988</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary</td>
			<td>Polymicro</td>
			<td>1068150019</td>
			<td>to make nanoflow column (75 &#181;m inner diameter x 360 &#181;m outer diameter)</td>
		</tr>
		<tr>
			<td>Cryostat cup</td>
			<td>Sigma-Aldrich</td>
			<td>E6032</td>
			<td>any cup or mold should work</td>
		</tr>
		<tr>
			<td>&#160;Microcentrifuge Tubes</td>
			<td>Eppendorf</td>
			<td>30108434</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde - D2</td>
			<td>Sigma-Aldrich</td>
			<td>492620</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid Optima LC/MS grade</td>
			<td>Fisher Chemical</td>
			<td>A117-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Difco</td>
			<td>214340</td>
			<td>place in 37 &#176;C water bath to melt</td>
		</tr>
		<tr>
			<td>Hydrophobic barrier pen</td>
			<td>Vector Labs</td>
			<td>15553953</td>
			<td></td>
		</tr>
		<tr>
			<td>Indium tin oxide (ITO)-coated glass slides</td>
			<td>Delta Technologies</td>
			<td>CB-90IN-S107</td>
			<td>25 mm x 75 mm x 0.8 mm (width x length x thickness)</td>
		</tr>
		<tr>
			<td>LC-MS vials</td>
			<td>Thermo</td>
			<td>TFMSCERT5000-30LVW</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol Optima LC/MS Grade</td>
			<td>Fisher Chemical</td>
			<td>A456-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma-Aldrich</td>
			<td>P7793</td>
			<td>Hydrophobic film</td>
		</tr>
		<tr>
			<td>pH-Indicator strips</td>
			<td>Supelco</td>
			<td>109450</td>
			<td></td>
		</tr>
		<tr>
			<td>Red phosphorus clusters</td>
			<td>Sigma-Aldrich</td>
			<td>343242</td>
			<td></td>
		</tr>
		<tr>
			<td>Reversed phase C18 material</td>
			<td>Waters</td>
			<td>186002350</td>
			<td>manually packed into nanoflow column</td>
		</tr>
		<tr>
			<td>Wite-out pen</td>
			<td>BIC</td>
			<td>150810</td>
			<td></td>
		</tr>
		<tr>
			<td>ZipTip</td>
			<td>Millipore</td>
			<td>Z720070</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments and Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic matrix sprayer system- M5</td>
			<td>HTX Technologies, LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge - 5424 R</td>
			<td>Eppendorf</td>
			<td>05-401-205</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat- HM 550</td>
			<td>Thermo Fisher Scientific</td>
			<td>956564A</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccant</td>
			<td>Drierite</td>
			<td>2088701</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>WPI</td>
			<td>501764</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI stainless steel target plate</td>
			<td>Bruker Daltonics</td>
			<td>8280781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite XLS</td>
			<td>Rainin</td>
			<td>17014391</td>
			<td>200 &#181;L</td>
		</tr>
		<tr>
			<td>Q Exactive Plus Hybrid Quadrupole-Orbitrap</td>
			<td>Thermo Fisher Scientific</td>
			<td>IQLAAEGAAPFALGMBDK</td>
			<td></td>
		</tr>
		<tr>
			<td>RapifleX MALDI-TOF/TOF</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac - SVC100</td>
			<td>Savant</td>
			<td>SVC-100D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Cleaner</td>
			<td>Bransonic</td>
			<td>2510R-MTH</td>
			<td>for sonication</td>
		</tr>
		<tr>
			<td>Ultrasonic homogenizer</td>
			<td>Fisher Scientific</td>
			<td>FB120110</td>
			<td>FB120 Sonic Dismembrator with CL-18 Probe</td>
		</tr>
		<tr>
			<td>Vaccum pump- Alcatel 2008 A</td>
			<td>Ideal Vacuum Products</td>
			<td>P10976</td>
			<td>ultimate pressure = 1 x 10-4 Torr</td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Corning</td>
			<td>6775</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath (37C) - Isotemp 110</td>
			<td>Fisher Scientific</td>
			<td>15-460-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Analysis Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Expasy</td>
			<td>https://web.expasy.org/translate/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlexAnalysis</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlexControl</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlexImaging</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PEAKS Studio</td>
			<td>Bioinformatics Solutions, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SCiLS Lab</td>
			<td>https://scils.de/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SignalP 5.0</td>
			<td>https://services.healthtech.dtu.dk/service.php?SignalP-5.0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tBLASTn</td>
			<td>http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&#38;BLAST_ 
			PROGRAMS=tblastn&#38;PAGE_ 
			TYPE=BlastSearch&#38;SHOW_ 
			DEFAULTS=on&#38;LINK_LOC 
			=blasthome</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

multi-faceted mass spectrometric investigation of neuropeptides in callinectes sapidus

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and response to external stressors. Yet, the biochemical mechanisms and full complement of neuropeptides and their functional roles remain poorly understood. Characterization of these endogenous peptides is hindered by the immense diversity within this class of signaling molecules. Additionally, neuropeptides are bioactive at concentrations 100x - 1000x lower than that of neurotransmitters and are prone to enzymatic degradation after synaptic release. Mass spectrometry (MS) is a highly sensitive analytical tool that can identify, quantify, and localize analytes without comprehensive a priori knowledge. It is well-suited for globally profiling neuropeptides and aiding in the discovery of novel peptides. Due to the low abundance and high chemical diversity of this class of peptides, several sample preparation methods, MS acquisition parameters, and data analysis strategies have been adapted from proteomics techniques to allow optimal neuropeptide characterization. Here, methods are described for isolating neuropeptides from complex biological tissues for sequence characterization, quantitation, and localization using liquid chromatography (LC)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS. A protocol for preparing a neuropeptide database from the blue crab, Callinectes sapidus, an organism without comprehensive genomic information, is included. These workflows can be adapted to study other classes of endogenous peptides in different species using a variety of instruments.

The workflow for sample preparation and MS analysis is depicted in Figure 1. After the dissection of neuronal tissue, homogenization, extraction, and desalting are performed to purify neuropeptide samples. If isotopic label-based quantification is desired, samples are then labeled and desalted once again. The resulting sample is analyzed through LC-MS/MS for neuropeptide identification and quantification.
Neuropeptides identified through the proteomics software sh...

Watch this Scientific Journal Video about Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus at JoVE.com

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus

Mass spectrometric characterization of neuropeptides provides sequence, quantitation, and localization information. This optimized workflow is not only useful for neuropeptide studies, but also other endogenous peptides. The protocols provided here describe sample preparation, MS acquisition, MS analysis, and database generation of neuropeptides using LC-ESI-MS, MALDI-MS spotting, and MALDI-MS imaging.

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and ...

The analysis of endogenous peptides, which are smaller than most proteins, but larger than many digested peptides is an under examined field. This protocol addresses the scap in mass spectrometry based workflows. Mass spectrometry is highly sensitive and it can be used to target specific neuropeptides of interest or can capture and detect a range of neuropeptides within a sample.The quantitation and localization of neuropeptide expression by mass spectrometry can lead to the development of peptide therapeutics and the discovery of neuropeptide biomarkers for various diseases. To begin the neuropeptide extraction collect brain tissue from the crustacean and using forceps immediately place one tissue each in a 0.6 milliliter tube containing 20 microliters of acidified methanol. Add 150 microliters of acidified methanol to the sample.Set the total Sonic patient time to 24 seconds, pulse time to eight seconds. Pause time to 15 seconds an amplitude to 50%on an ultrasonic homogenizer and homogenize the samples on ice. Centrifuge the sample at four degrees Celsius at 20, 000 GS for 20 minutes.With a pipette transferred the supernatant to a tube and dry it in a vacuum concentrator at approximately 35 degree Celsius. For desalting reconstitute the extracted tissue sample in 20 microliters of 0.1%formic acid. Vortex the solution and sonicate in a room temperature water bath for one minute.Apply 0.5 microliters of sample to a pH strip to confirm that the pH is less than four. Centrifuge at four degree Celsius and 20, 000 GS for 30 seconds. Prepare wetting equilibration wash in illusion solutions.Obtain a 10 microliter desalting tip with C18 resin. Place the desalting tip on a 20 microliter pipette that is set to 15 microliters. Aspirate the tip three times with wetting solution and three times with equi collaboration solution.Aspirate in the sample 10 times followed by washing three times in wash solution discarding each wash. Elute by aspirating 10 times in each of the illusion solutions in order of increasing Acetonitrile. Dry the alluded neuropeptides in a vacuum concentrator.Reconstitute the dried desalted neuropeptides in five microliters of 0.1%formic acid. Vortex the solution and sonicate it in a room temperature water bath for one minute. Briefly centrifuge at 2000 GS.For spotting of neuropeptides and crusta tissues, pipette a three microliter droplet of sample onto a hydrophobic film.Pipette three microliters of DHB matrix directly on the sample droplet. Mix the solutions by pipetting up and down. Pipette one microliter of the sample and matrix mixture into a well of the MALDI stainless steel target plate and use the pipette tip to spread each mixture out to the edges of the sample well.Spot one microliter of one-to-one caliber and matrix mixture into a well near the sample. Insert target plate containing dried sample spots into MALDI tandem time-of-flight instrument. With the DHB matrix set laser power to 95%Select automatic optimal detector gain and smart complete sample sample carrier movement mode.Acquire MS Spectra in the range of 200 to 3, 200 M over Z and add multiple spectra from each spot together to increase neuropeptide signal to noise ratio. Fill half a cryostat cup with gelatin and allow it to solidify at room temperature. Keep leftover liquid gelatin warm in a 37 degree Celsius water bath.Collect desired neuronal tissue from the animal and use forceps to immediately dip the tissue into a 0.6 milliliter tube containing deionized water for one second. Place the tissue on top of the solid gelatin and fill the rest of the cryostat cup with liquid gelatin. Use forceps to position the tissue.Place the cryostat cup on a flat surface and freeze with dry ice. For sectioning preparation separate the gelatin embedded sample from the cryostat mold by cutting the mold away. Mount the embedded tissue onto a cryostat chuck by pipetting a one milliliter droplet of deionized water onto the chuck and immediately pressing the embedded tissue onto the droplet.Once frozen pipette, more deionized water around the tissue to further secure it to the chuck. Section the tissue at an approximate thickness of one cell. Thumb mount each section onto an Indium tin oxide coated glass slide by placing one side of the slide near the section and placing a finger on the other side of the slide to slowly warm the glass and allow the section to stick to the slide.Export De Novo only peptides. CSV from peaks with an average local confidence score of greater than or equal to 75. Search the peptide list for known sequence motifs indicative of neuropeptides belonging to specific neuropeptide families.Choose a known pre-pro hormone amino acid sequence of interest and use TB blast N to search query pre-pro hormone sequence against a database. Select the target organism and change expect threshold algorithm parameters to 1000 to include low score alignments. Run blast program and then check the results for high homology scores between query and subject sequences producing significant alignments.Save the FASTA file containing nucleotide sequence. Translate pre-pro hormone nucleotide sequence into pre-pro hormone peptide sequences using Expasy translate tool. For callinectes sapidus select invertebrate mitochondrial for genetic code and run the tool.Check for signal peptide sequence and pro hormone cleavage sites in the peptide sequences using signal P.This is a fragmentation spectrum of a neuropeptide identified with 100%sequence coverage in a low fragment mass error with a maximum limit of 0.02 Dalton. Here is a spectrum of a neuropeptide also identified with 100%sequence coverage, but with a low number of fragment ions, therefore the confidence of the identification is low. These are the extracted ion chromatograms for a single neuropeptide that was identified in two separate and consecutive runs.Even though the retention time differs slightly, this can still be used for label free quantification because the time shift is less than one minute. This is an example of a full scan mass spectrum containing a neuropeptide that was Dimethyl labeled resulting in a 4.025 Dalton mass shift between the light and heavy forms of the neuropeptide. The fragment ion mass spectrum of a De Novo sequenced peptide demonstrates good fragmentation coverage, where B and or Y fragment ion was observed for each amino acid with a low mass error of 0.02 Dalton.This indicates that an endogenous peptide belonging to the crustacean RYMI neuropeptide family was observed. Examples of good spots that touch all edges of the well engraving are shown on the left. And examples of bad spots are shown on the right.After MALDI MS imaging of neuropeptides from callinectes sapidus tissues, ion distribution images of various M over Z values were obtained corresponding to different neuropeptides. Extraction solvent optimization for a particular analy type is crucial. Also ensure complete homogenization occurs and adjust the method as needed.Nothing will be detected downstream if it isn't extracted. This technique can provide a foundational list of important neuropeptides that can be further investigated as potential biomarkers, as well as provide untargeted localization information without the need for antibodies.

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