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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;10 cm, ID75 &#181;m, 3 &#181;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-13C, d2, 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 &#181;m, 5 &#181;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&#160;&#160; (Oasis HLB 1 cc Cartridge)</td>
			<td>Waters</td>
			<td>186000383</td>
			<td>Oasis HLB 1 cc Cartridge</td>
		</tr>
		<tr>
			<td>Sodium cyanoborodeuteride - NaBD3CN</td>
			<td>Sigma-Aldrich</td>
			<td>190020</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cyanoborohydride - NaBH3CN</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 Na2HPO3 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

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.

We present an optimized tandem mass tag (TMT) labeling protocol that includes detailed information for each of the following steps: protein extraction, quantification, precipitation, digestion, labeling, submission to a proteomics facility, and data analyses.

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which are involved in many cell activities. Here, we describe a rapid and reliable method for the absolute qualification of nucleoside/nucleotide contents in plants. Briefly, 100 mg of homogenized plant material was extracted with 1 mL of extraction buffer (methanol, acetonitrile, and water at a ratio of 2:2:1). Later, the sample was concentrated five times in a freeze dryer and then injected into an HPLC-MS/MS. Nucleotides were separated on a porous graphitic carbon (PGC) column and nucleosides were separated on a C18 column. The mass transitions of each nucleoside and nucleotide were monitored by mass spectrometry. The contents of the nucleosides and nucleotides were quantified against their external standards (ESTDs). Using this method, therefore, researchers can easily quantify nucleosides/nucleotides in different plants.

A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise.
Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. 
We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines.

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

Large-Scale Multi-Omics Genome-Wide Association Studies (Mo-GWAS): Guidelines for Sample Preparation and Normalization

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect and quantify hundreds of thousands of metabolite features. However, the application of these techniques to a large number of samples is subject to more complex interactions, particularly for genome-wide association studies (GWAS). This protocol describes an optimized metabolic workflow, which combines an efficient and fast sample preparation with the analysis of a large number of samples for legume crop species. This slightly modified extraction method was initially developed for the analysis of plant and animal tissues and is based on extraction in methyl tert-butyl ether: methanol solvent to allow the capture of polar and lipid metabolites. In addition, we provide a step-by-step guide for reducing analytical variations, which are essential for the high-throughput evaluation of metabolic variance in GWAS.

Large-Scale Multi-Omics Genome-Wide Association Studies Mo-GWAS: Guidelines for Sample Preparation and Normalization

In this protocol, we present an optimized workflow, which combines an efficient and fast sample preparation of many samples. In addition, we provide a step-by-step guide to reduce analytical variations for high-throughput evaluation of metabolic GWAS studies.

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect ...

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown into highly ordered two-dimensional (2D) crystals under favorable conditions. The quality of the grown 2D crystals is crucial to the resolution of the final reconstruction via 2D image processing. Over the years, lipid monolayers have been used as a supporting layer to foster the 2D crystallization of peripheral membrane proteins as well as soluble proteins. This method can also be applied to 2D crystallization of integral membrane proteins but requires more extensive empirical investigation to determine detergent and dialysis conditions to promote partitioning to the monolayer. A lipid monolayer forms at the air-water interface such that the polar lipid head groups remain hydrated in the aqueous phase and the non-polar, acyl chains, tails partition into the air, breaking the surface tension and flattening the water surface. The charged nature or distinctive chemical moieties of the head groups provide affinity for proteins in solution, promoting binding for 2D array formation. A newly formed monolayer with the 2D array can be readily transfer into an electron microscope (EM) on a carbon-coated copper grid used to lift and support the crystalline array. In this work, we describe a lipid monolayer methodology for cryogenic electron microscopic (cryo-EM) imaging.

Lipid monolayers have been used as a foundation for forming two-dimensional (2D) protein crystals for structural studies for decades. They are stable at the air-water interface and can serve as a thin supporting material for electron imaging. Here we present the proven steps on preparing lipid monolayers for biological studies.

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown ...