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.
