The development and use of the techniques described here were supported by the Brazilian National Research Council grant 420811/2018-4 (LMC); Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (www.fapesp.br) grants 2019/16023-6 (LMC), 2019/17433-3 (LOF) and 21/01286-1 (MEME). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the article.

The following procedure for peptide extraction and reductive methylation was adapted from previously published procedures24,25,26,27. This protocol followed the guidelines of the National Council for Animal Experimentation Control (CONCEA) and was approved by the Ethics Commission for Animal Use (CEUA) at Bioscience Institute of Sao Paulo State University. The protocol steps are shown in Figure 1.
NOTE: Prepare all aqueous solutions in ultrapure water.
1. Peptide extraction
<ol>
	<li>Cell culture
	<ol>
		<li>Cultivate SHSY5Y cells in a 15 cm dish at 37 &#176;C under 5% CO2 in Dulbecco&#39;s modified Eagle&#39;s medium containing 15% fetal bovine serum and 1% Penicillin-Streptomycin.</li>
		<li>Use 2-3 plates for each sample. Grow the cells to 100% confluence.</li>
		<li>After treatment intended, wash the cells twice with phosphate-buffered saline. Next, add 10 mL of Phosphate-buffered saline, scrape the cells and collect into a 15 mL tube.</li>
		<li>Centrifugate at 800 x g for 5 min and remove the supernatant. Resuspend the pellet in 1 mL of ultra-purified deionized water at 80 &#176;C.</li>
		<li>Transfer the contents of the tube (cellular lysate) to a 2 mL microfuge tube.</li>
	</ol>
	</li>
	<li>Animal tissues (mainly nervous tissue):
	<ol>
		<li>Anesthetize wild-type male adult Danio rerio (zebrafish) with a lethal dose of MS 222 (100 mg/L) and immediately subject it to 8 s of microwave radiation to inactivate peptidase and protease. 
		NOTE: A household-type microwave oven can be used. A 900 W microwave was used for 8 to 10 s at full power. The microwave used must be able to raise the brain temperature to &#62; 80 &#176;C within 10 s. Reproducibility in heating between samples would also be benefited by placing the tissue in the same location in the microwave.</li>
		<li>After heat-inactivation, collect the whole brain in a 2 mL microfuge tube and freeze at &#8722;80 &#176;C until analysis.</li>
		<li>Resuspend the tissue sample in 1 mL of ultra-purified deionized water at 80 &#176;C. Sonicate the tissue with a probe using 30 pulses (4 Hz) of 1 s. 
		NOTES: For liver and kidney tissues, use a mechanical homogenizer at 10,000-30,000 rpm for 20 s. For muscular tissues, grind the tissue in liquid nitrogen using a porcelain crucible and pestle. The following steps are the same for the cellular lysate or the homogenate tissue.</li>
	</ol>
	</li>
	<li>Incubate the cellular lysate or homogenate tissue at 80 &#176;C for 20 min. Next, cool it on ice for 10-30 min.</li>
	<li>Add 10 &#181;L of 1 M HCl stock solution for each 1 mL of sample volume to obtain a final concentration of 10 mM. Mix by vortexing for 20 s and further incubate on ice for 15 min. 
	NOTE: Before acidifying, ensure the sample is completely cooled to avoid breaking the peptide bonds caused by acidification at elevated temperatures.</li>
	<li>Centrifuge the cellular lysate or the homogenate tissue at 12,000 x g at 4 &#176;C for 15 min. Collect the supernatant in low binding protein microcentrifuge tubes and store it at -80 &#176;C.</li>
	<li>Clean the ultrafiltration devices (10 kDa cut-off filters) by adding water and centrifuge at 2300 x g for 3 min. Repeat this step two more times.</li>
	<li>Place the supernatant in the pre-washed 10 kDa cut-off filters and centrifuge at 2,300 x g at 4 &#176;C for 50 min in a refridgerated centrifuge. The flow-through represents the peptide extract.</li>
	<li>Desalt the samples on reversed-phase cleanup columns according to the manufacturer&#39;s instructions using acetonitrile (ACN) and trifluoroacetic acid (TFA) solutions as described below:
	<ol>
		<li>Equilibrate the column with 1 mL of 100% ACN.</li>
		<li>Wash the column with 1 mL solution of 5% ACN with 0.1% TFA.</li>
		<li>Load the complete volume of the sample in the column.</li>
		<li>Wash the column with 1 mL solution of 5% ACN with 0.1% TFA</li>
		<li>Elute the peptides from the column with a 1.8 mL solution of 100% ACN with 0.15% TFA in protein low binding microcentrifuge tubes.</li>
	</ol>
	</li>
	<li>Dry the sample completely in a vacuum centrifuge. Set the concentration method for organic solvents and temperature at 30 &#176;C. The concentration time is monitored on display.
	<ol>
		<li>Store the samples at &#8722;80&#176;C until the next step.</li>
	</ol>
	</li>
</ol>
2. Peptide quantification with fluorescamine
NOTE: The amount of peptide can be estimated using fluorescamine at pH 6.8 as previously described11,28. This method consists of the attachment of a fluorescamine molecule to the primary amines present in the lysine (K) residues and/or the N-terminal of peptides. The reaction is performed at pH 6.8 to guarantee that the fluorescamine reacts only with the amino groups of the peptides and not with free amino acids. The fluorescamine is measured by using a spectrofluorometer at an excitation wavelength of 370 nm and an emission wavelength of 480 nm.
<ol>
	<li>Prepare different concentrations of the standard peptide (0.05, 0.1, 0.15, 0.2, 0.3, 0.5 and 0.7 &#181;g/&#181;L) and store the aliquots at -20 &#176;C. 
	NOTE: The peptide 5A (LTLRTKL) is suggested since it has a known composition and concentration.</li>
	<li>Prepare fluorescamine stock solution (0.3 mg/mL) in acetone. Aliquot quickly in microcentrifuge tubes (1 mL), seal using parafilm, and store at -20 &#176;C in the dark.</li>
	<li>Prepare 0.2 M Phosphate buffer (PB) at pH 6.8. 
	NOTE: Prepare 0.2 M PB by adding 0.1 M phosphate buffer pH 6.8 (26.85 mL of Na2HPO3 1M) and 0.1 M phosphate buffer pH 6.8 (23.15 mL of NaH2PO3 1M) to 250 mL of water</li>
	<li>Resuspend the peptide samples in 100-200 &#181;L of ultra-purified water.</li>
	<li>Pipette 2.5 &#181;L of the standard peptide concentrations and samples onto the white 96-well plate for fluorescence assays in triplicate. Add 25 &#181;L of 0.2 M Phosphate buffer.</li>
	<li>Add 12.5 &#181;L of fluorescamine with a multichannel pipette. Homogenize gently for 1 min on the orbital rotator shaker.</li>
	<li>Next, add 110 &#181;L of water with a multichannel pipette to stop the reaction. 
	NOTE: Transfer the fluorescamine stock solution and ultrapure water to two reservoirs to pipette these solutions with a multichannel pipette.</li>
	<li>Adjust the following reading parameters on the spectrofluorometer: Read the samples from the top, excitation wavelength at 370 nm, and emission wavelength at 480 nm.</li>
	<li>Read the plate on the spectrofluorometer.</li>
</ol>
3. Reductive methylation of amines labeling
NOTE: This isotopic labeling method is based on the dimethylation of amine groups with deuterated and non-deuterated forms of formaldehyde and sodium cyanoborohydride reagents. The final product of this reaction adds 28 Da, 30 Da, 32 Da, 34 Da, or 36 Da to the final mass of each peptide at each available labeling site (lysine or N-terminal). This reaction produces an m/z difference in the peptides labeled with different forms observed in the MS spectrum (Table 1).
CAUTION: Proper safety equipment should be used to handle these compounds, and care should be taken to minimize exposure. Procedures with formaldehyde and sodium cyanoborohydride reagents should be performed in a fume hood because they are very toxic (including weighing the sodium cyanoborohydride). During the quenching reaction and acidification, a toxic gas (hydrogen cyanide) may be generated.
<ol>
	<li>Prepare the following fresh solutions from the stock or reagents on the day of the procedure in ultrapure water:
	<ol>
		<li>Dilute the stock of 37% Formaldehyde (CH2O) to 4%.</li>
		<li>Dilute the stock of 20% Formaldehyde Deuterated (CD2O) to 4%.</li>
		<li>Dilute the stock of 20 % Deuterated C13 Formaldehyde (13CD2O) to 4%.</li>
		<li>Prepare 0.6 M NaBH3CN.</li>
		<li>Prepare 0.6 M NaBD3CN.</li>
		<li>Prepare a solution of 1% Ammonium bicarbonate.</li>
		<li>Prepare a solution 5% Formic acid. 
		NOTE: Regarding the relative quantitation of peptides, as different experimental schemes can be performed depending on the number of labels used, attention is necessary during the chemical labeling procedure. It is recommended to separate small aliquots of the labels in separate racks with the respective samples to be labeled to reduce the potential for human error in adding the wrong reagent to the sample tubes.</li>
	</ol>
	</li>
	<li>Prepare each sample containing up to 25 &#181;g of the peptide. Samples must not contain Tris or Ammonium Bicarbonate. 
	NOTE: The quantities described below are sufficient for each sample in a volume of 100 &#181;L. 
	Proceed with steps 3.3-3.8 in a fume hood.</li>
	<li>Add 1/10th volume of 1 M TEAB to the samples (final concentration of the solution 100 mM of TEAB). Check the pH with a pH indicator paper; it must be between 5-8. Adjust with HCl or NaOH if needed.</li>
	<li>Add 4 &#181;L of non-deuterated, deuterated Formaldehyde or C13 deuterated Formaldehyde according to the established labeling scheme. Mix for 5 s by vortexing.</li>
	<li>Add 4 &#181;L of NaBH3CN (0.6 M) or NaBD3CN (0.6 M) according to the established labeling scheme. Mix for 5 s by vortexing.</li>
	<li>Incubate in a fume hood for 2 h at room temperature, mixing every 30 min.</li>
	<li>Repeat steps 3.4 and 3.5. Incubate the samples in a fume hood overnight at room temperature.</li>
	<li>Add 16 &#181;L of Ammonium bicarbonate (1%) and mix by vortexing. Place the sample on ice, add 8 &#181;L of formic acid (5%), and mix by vortex for 5 s.</li>
	<li>Combine the samples, adjust the pH to 2-4 and desalt the combined samples on reversed-phase cleanup columns as previously described in step 1.8.</li>
	<li>Dry the sample completely in a vacuum centrifuge. Set the concentration method for organic solvents and temperature at 30 &#176;C. The concentration time is monitored on display.</li>
	<li>Store the samples at -20 &#176;C.</li>
</ol>
4. Liquid chromatography and mass spectrometry
<ol>
	<li>Perform LC-MS analysis using a nanoHPLC system coupled to a MS instrument compatible with methyl tags. 
	&#8203;NOTE: A variety of MS instruments are compatible with RMA labeling.A nanoHPLC system coupled to Orbitrap is typically used to perform LC-MS analysis through a nanoelectrospray ion source. First, the sample is loaded into a precolumn and peptides separated in an analytical column. The elution of peptides is performed using a linear gradient of 5%-45% acetonitrile, in 0.1% formic acid, during 90 min, with a flow of 200 nL/min. The mass spectrometer is set to function in data-dependent mode. Each full scan is acquired at an intensity of 10-30 eV, 2.3 Kv, and then the ten highest peaks are selected for collision-induced dissociation (CID) fragmentation. The injection time is set on the ion trap at 100 ms, and the Fourier Transform (FT)-MS injection is fixed with a resolution of 1000 ms 30,000 at m/z 300-1800. A minimum of 5000 counts and a dynamic exclusion of 70 s is used to perform fragmentation scanning.</li>
</ol>
5. Relative quantitation of peptides
NOTE: The MS spectra are analyzed in the mass spectrometer software. Peak groups of labeled peptides with different tags are identified in the MS spectra. The relative quantitation is calculated by the intensity of each monoisotopic peak. Each treated group is compared to the respective control group.
<ol>
	<li>Right double-click on the raw sample file to open the spectrum analysis software. Load the Retention time (RT) and MS spectrum (EM) chromatograms in two tabs, top and bottom, respectively.</li>
	<li>Right-click once sequentially on the Display and Mass options icons in the software toolbar and set the mass precision to four decimals.</li>
	<li>Position the mouse cursor anywhere on the RT tab. Look for the retention time of the corresponding ion to be analyzed and click the right mouse button. The MS spectrum of the selected time will automatically be shown in the EM tab.</li>
	<li>Position the mouse cursor anywhere on the EM tab. Look for the ions to be analyzed.</li>
	<li>Right-click and hold in an adjacent region to the left near these ions. Then, drag the mouse to the right at the desired range to zoom the region of interest.</li>
	<li>Keep the mouse positioned on the EM tab and click on the right or left keyboard arrows to define the range of ions to be analyzed.</li>
	<li>Position the mouse cursor the RT tab again at the beginning of the desired time interval.</li>
	<li>Right-click and drag the mouse until the chosen time value. Leave the button. The accumulated intensity of the ions will automatically be shown on the EM tab.</li>
	<li>Collect the m/z, z, and ion intensity data on a spreadsheet. 
	NOTE: The monoisotopic mass of each peptide without added methyl groups is calculated from the following formula: 
	 
	Mass unmodified peptide = (m/z a x z) - (C a x T) - (1.008 x z) 
	 
	m/za is the observed mass to charge value for the monoisotopic peak for each peptide labeled with different combinations of tags (a =1, 2, 3, 4 or 5, corresponding to the sample number). 
	z is charge state. 
	Ca&#160;is the monoisotopic mass of a pair of methyl groups: 
	For a=1, Ca = 28.0313 (the net addition of two CH3 groups to the primary amine) 
	For a=2, Ca = 30.0439 for two CHD2 groups 
	For a=3, Ca = 32.0564 for two CD2H groups 
	For a=4, Ca= 34.0690 for two CD3 groups 
	For a=5, Ca = 36.0757 for two 13 CD3 groups 
	T is the number of pairs of methyl groups incorporated into the peptide. This can be calculated from the following formula when five tags are used: T=z*(m/z5 - m/z1)/8. For peptides that contain a single primary amine and therefore are labeled with only two methyl groups present peak overlaps on the MS spectra when adjacent labels are used. The peak intensity of each labeled peptide can be corrected using the equations described by Tashima and Fricker25.</li>
</ol>
6. Peptide Identification
<ol>
	<li>To identify peptides, analyze the MS/MS data using a database search engine29,30.</li>
	<li>To calculate the false discovery rate (FDR) using the decoy fusion method, search a decoy database. 
	&#8203;NOTE: The search parameters generally used are no enzyme specificity; precursor mass tolerance set 15-50 ppm; fragment ion mass tolerance of 0.5 Da; variable modifications: reactive amines from Lys residues and N-terminus of the peptides isotopic methylated labels (L1 (+28), L2 (+30), L3 (+32), L4 (+34) and L5 (+36)), oxidized methionine (+15.99 Da) and acetylation (+42.01 Da).</li>
	<li>Then, sort the peptides by their average of local confidence to select the best spectra to annotate and filter them by FDR &#8804;5%.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62971/62971fig01.jpg" /> 
Figure 1: Peptidomic studies workflow. Steps of peptide extraction and Reductive Methylation of amines. <a href="https://www.jove.com/files/ftp_upload/62971/62971fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidomic+approaches+to+study+proteolytic+activity.">Peptidomic approaches to study proteolytic activity.</a> Current Protocols in Protein Science. , 13 (2011).</li><li>Udenfriend, S., 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=Fluorescamine:+a+reagent+for+assay+of+amino+acids,+peptides,+proteins,+and+primary+amines+in+the+picomole+range.">Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range.</a> Science. 178 (4063), 871-872 (1972).</li><li>Ma, B., 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=PEAKS:+powerful+software+for+peptide+de+novo+sequencing+by+tandem+mass+spectrometry.">PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 17 (20), 2337-2342 (2003).</li><li>Zhang, J., 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=PEAKS+DB:+de+novo+sequencing+assisted+database+search+for+sensitive+and+accurate+peptide+identification.">PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification.</a> Molecular &amp; Cellular Proteomics. 11 (4), 1-8 (2012).</li><li>Sturm, R. M., Dowell, J. A., 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=Rat+brain+neuropeptidomics:+tissue+collection,+protease+inhibition,+neuropeptide+extraction,+and+mass+spectrometric+analysis.">Rat brain neuropeptidomics: tissue collection, protease inhibition, neuropeptide extraction, and mass spectrometric analysis.</a> Methods in Molecular Biology. 615, 217-226 (2010).</li><li>Fricker, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+mass+spectrometry-based+peptidomic+approaches.">Limitations of mass spectrometry-based peptidomic approaches.</a> Journal of the American Society for Mass Spectrometry. 26 (12), 1981-1991 (2015).</li><li>Ross, , 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=Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents.">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Molecular &amp; Cellular Proteomics. 3, 1154-1169 (2004).</li><li>Thompson, 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=Tandem+mass+tags:+a+novel+quantification+strategy+for+comparative+analysis+of+complex+protein+mixtures+by+MS/MS.">Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.</a> Analytical Chemistry. 75, 1895-1904 (2003).</li></ol>

No competing financial interests exist.

In most peptidomics studies, one of the critical steps is, without doubt, the sample preparation that should be carefully performed to avoid the presence of peptide fragments generated by proteases after a few minutes post-mortem. The initial studies on brain extracts prepared from non-microwaved samples showed a large number of protein fragments to be present in the 10-kDa microfiltrates. Different approaches have been described to avoid peptide spectra from protein degradation: focused microwave irradiation animal sacr...

&#34;Omics&#34; sciences are characterized by the deep analysis of a molecule set, such as DNA, RNA, proteins, peptides, metabolites, etc. These generated large-scale datasets (genomics, transcriptomics, proteomics, peptidomics, metabolomics, etc.) have revolutionized biology and led to an advanced understanding of biological processes1. The term peptidomics began to be introduced in the early 20th century, and some authors have referred to it as a branch of proteomics2. However, peptidomics has distinct particularities, where the main interest is to investigate the naturally generated peptides content during cellular processes, as well as the characterization of biological activity of these molecules3,4.
Initially, bioactive peptide studies were restricted to the neuropeptides and hormone peptides through Edman degradation and radioimmunoassay. However, these techniques do not allow a global analysis, depending on the isolation of each peptide in high concentrations, time for the generation of antibodies, besides cross-reactivity possibility5.
Peptidomics analysis was only made possible after several advances in Liquid chromatography coupled mass spectrometry (LC-MS) and genome projects that delivered comprehensive data pools for proteomics/peptidomics studies6,7. Moreover, a specific peptide extraction protocol for peptidomes needed to be established because the first studies that analyzed neuropeptides globally in brain samples showed that detection was affected by the massive degradation of proteins, which occur mainly in this tissue after 1 min post-mortem. The presence of these peptide fragments masked the neuropeptide signal and did not represent the peptidome in vivo. This problem was solved mainly with the application of fast heating inactivation of proteases using microwave irradiation, which drastically reduced the presence of these artifact fragments and allowed not only the identification of neuropeptide fragments but revealed the presence of a set of peptides from cytosolic, mitochondrial, and nuclear proteins, different of degradome6,8,9.
These methodological procedures allowed an expansion of the peptidome beyond the well-known neuropeptides, where hundreds of intracellular peptides generated mainly by the action of proteasomes have been identified in yeast10, zebrafish11, rodent tissues12, and human cells13. Dozens of these intracellular peptides have been extensively shown to have both biological and pharmacological activities14,15. Furthermore, these peptides can be used as disease biomarkers and possibly have clinical significance, as demonstrated in cerebrospinal fluid from patients with intracranial saccular aneurysms16.
Currently, in addition to the identification of peptide sequences, it is possible through mass spectrometry to obtain data of absolute and relative quantitation. In the absolute quantitation, the peptide levels in a biological sample are compared to synthetic standards, while in the relative quantitation, the peptide levels are compared among two or more samples17. Relative quantitation can be performed using the following approaches: 1) &#34;label free&#34;18; 2) in vivo metabolic labeling or 3) chemical labeling. The last two are based on the use of stable isotopic forms incorporated into peptides19,20. In label-free analysis, the peptide levels are estimated by considering the signal strength (spectral counts) during the LC-MS18. However, isotopic labeling can obtain more accurate relative levels of peptides.
Many peptidomic studies used trimethylammonium butyrate (TMAB) labeling reagents as chemical labeling, and more recently, Reductive Methylation of Amines (RMA) with deuterated and non-deuterated forms of formaldehyde and sodium cyanoborohydride reagents have been used11,21,22. However, the TMAB labels are not commercially available, and the synthesis process is very laborious. On the other hand, in the RMA, the reagents are commercially available, inexpensive compared to other labels, the procedure is simple to perform, and the labeled peptides are stable23,24.
The use of RMA involves forming a Schiff base by allowing the peptides to react with formaldehyde, followed by a reduction reaction through the cyanoborohydride. This reaction causes dimethylation of free amino groups on N-terminals and lysine side chains and monomethylates N-terminal prolines. How proline residues are often rare on the N-terminal, practically all peptides with free amines on the N-terminus are labeled with two methyl groups23,24,25.

<table><tbody><tr><td>10 kDa cut-off filters</td><td>Merck Millipore</td><td>UFC801024</td><td>Amicon Ultra-4, PLGC Ultracel-PL Membrane, 10 kDa</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>179124</td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>1000291000</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma-Aldrich</td><td>11213</td><td></td></tr><tr><td>analytical column (EASY-Column)</td><td>EASY-Column</td><td>(SC200)</td><td>&amp;nbsp;10 cm, ID75 &amp;micro;m, 3 &amp;micro;m, C18-A2</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate</td><td>Sigma-Aldrich</td><td>E10521</td><td>MS-222</td></tr><tr><td>Fluorescamine</td><td>Sigma-Aldrich</td><td>F9015</td><td></td></tr><tr><td>Formaldehyde solution</td><td>Sigma-Aldrich</td><td>252549</td><td></td></tr><tr><td>Formaldehyde-&lt;sup&gt;13&lt;/sup&gt;C, d&lt;sub&gt;2&lt;/sub&gt;, solution</td><td>Sigma-Aldrich</td><td>596388</td><td></td></tr><tr><td>Formaldehyde-d2 solution</td><td>Sigma-Aldrich</td><td>492620</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>33015</td><td></td></tr><tr><td>Fume hood</td><td>Quimis</td><td>Q216</td><td></td></tr><tr><td>Hydrochloric acid - HCl</td><td>Sigma-Aldrich</td><td>258148</td><td></td></tr><tr><td>LoBind-Protein retention tubes</td><td>Eppendorf</td><td>EP0030108116-100EA</td><td></td></tr><tr><td>LTQ-Orbitrap Velos</td><td>Thermo Fisher Scientific</td><td>LTQ Velos</td><td></td></tr><tr><td>Microwave oven</td><td>Panasonic</td><td>NN-ST67HSRU</td><td></td></tr><tr><td>n Easy-nLC II nanoHPLC</td><td>Thermo Fisher Scientific</td><td>LC140</td><td></td></tr><tr><td>PEAKS Studio</td><td>Bioinformatics Solutions Inc.</td><td>VERSION 8.5</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>Invitrogen</td><td>3002</td><td>tablets</td></tr><tr><td>precolumn (EASY-Column)</td><td>Thermo Fisher Scientific</td><td>(SC001)</td><td>2 cm, ID100 &amp;micro;m, 5 &amp;micro;m, C18-A1</td></tr><tr><td>Refrigerated centrifuge</td><td>Hermle</td><td>Z326K</td><td>for conical tubes</td></tr><tr><td>Refrigerated centrifuge</td><td>Vision</td><td>VS15000CFNII</td><td>for microtubes</td></tr><tr><td>Reversed-phase cleanup columns&amp;nbsp;&amp;nbsp; (Oasis HLB 1 cc Cartridge)</td><td>Waters</td><td>186000383</td><td>Oasis HLB 1 cc Cartridge</td></tr><tr><td>Sodium cyanoborodeuteride - NaBD&lt;sub&gt;3&lt;/sub&gt;CN</td><td>Sigma-Aldrich</td><td>190020</td><td></td></tr><tr><td>Sodium cyanoborohydride - NaBH&lt;sub&gt;3&lt;/sub&gt;CN</td><td>Sigma-Aldrich</td><td>156159</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>S9763</td><td>NOTE: 0.2 M PB= 0.1 M phosphate buffer pH 6.8 (26.85 mL of Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;3&lt;/sub&gt; 1M) plus 0.1 M phosphate buffer pH 6.8 (23.15 mL of NaH2PO3 1M) to 250 ml of water</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich</td><td>S3139</td><td></td></tr><tr><td>Sonicator</td><td>Qsonica</td><td>Q55-110</td><td></td></tr><tr><td>Standard peptide</td><td>Proteimax</td><td></td><td>amino acid sequence: LTLRTKL</td></tr><tr><td>Triethylammonium buffer - TEAB 1 M</td><td>Sigma-Aldrich</td><td>T7408</td><td></td></tr><tr><td>Trifluoroacetic acid - TFA</td><td>Sigma-Aldrich</td><td>T6508</td><td></td></tr><tr><td>Ultra purified water</td><td>Milli-Q</td><td>Direct-Q 3UV</td><td></td></tr><tr><td>Vacuum centrifuge</td><td>GeneVac</td><td>MiVac DNA concentrator</td><td></td></tr><tr><td>Water bath</td><td>Cientec</td><td>266</td><td></td></tr><tr><td>Xcalibur Software</td><td>ThermoFisher Scientific</td><td>OPTON-30965</td><td></td></tr></tbody></table>

sample preparation and relative quantitation using reductive methylation of amines for peptidomics studies

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

The results obtained from the runs carried out on the mass spectrometer are stored in raw data files that can be opened in the mass spectrometer software. In the MS spectra, it is possible to observe peak groups representing labeled peptides according to the labeling scheme used, ranging from 2-5 labels. For example, in Figure 2, pairs of peaks detected in a chromatographic time are represented in an experiment where only two isotopic labels were used in two different samples in the same run...

Watch this Scientific Journal Video about Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies at JoVE.com

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the ...

sample-preparation-relative-quantitation-using-reductive-methylation

Research

JoVE Journal

Biochemistry

2.2K Views.  Sao Paulo State University (UNESP). This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS. 

Video: Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive 13C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive 13C-methylation is a chemical reaction performed under mild conditions that modifies a protein&#39;s primary amino groups (lysine &#949;-amino groups and the N-terminal &#945;-amino group) to 13C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein 13C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of 13C-labeling at each primary amino group. One limitation of the method arises when the protein&#39;s N-terminal residue is a lysine because the &#945;- and &#949;-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the 13C-dimethylamines associated with both the N-terminal &#945;-amine and the side chain &#949;-amine. The NMR signals of the N-terminal &#945;-dimethylamine and the side chain &#949;-dimethylamine of hen egg white lysozyme, Lys1, are identified in 1H-13C heteronuclear single-quantum coherence spectra.

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Two methods for assigning the &#945;- and &#949;-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic ...

Chemistry

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer&#39;s disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls.

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in human pathology and other biochemical contexts. In order to perform characterization of protein deamidation, we have recently developed a novel long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method which can separate the glutamine (Gln) and asparagine (Asn) isoform products of deamidation from model compounds to highly complex biological samples. LERLIC-MS/MS is, therefore, the first shotgun proteomics strategy for the separation and quantification of Gln deamidation isoforms. We also demonstrate, as a novelty, that the sample processing protocol outlined here stabilizes the succinimide intermediate allowing its characterization by LERLIC-MS/MS. Application of LERLIC-MS/MS as shown in this video article can help to elucidate the currently unknown molecular arrays of protein deamidation. Additionally, LERLIC-MS/MS provides further understanding of the enzymatic reactions that encompass deamidation in distinct biological backgrounds.

Here we present a step-by-step protocol of the long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method. This is a novel methodology that enables for the first time quantification and characterization of the glutamine and asparagine deamidation isoforms by shotgun proteomics.

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in ...

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Neuroscience

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Cancer Research

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...