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 <stron...

<ol>
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C., 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=Current+peptidomics:+applications,+purification,+identification,+quantification,+and+functional+analysis.">Current peptidomics: applications, purification, identification, quantification, and functional analysis.</a> Proteomics. 15 (5-6), 1026-1038 (2015).</li><li>Chard, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+radioimmunoassay+and+related+techniques+(3rd+Ed).">An introduction to radioimmunoassay and related techniques (3rd Ed).</a> FEBS Letters. 238 (1), 223 (1988).</li><li>Svensson, M., Sk&#246;ld, K., Svenningsson, P., Andren, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidomics-based+discovery+of+novel+neuropeptides.">Peptidomics-based discovery of novel neuropeptides.</a> Journal of Proteome Research. 2 (2), 213-219 (2003).</li><li>Baggerman, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidomics.">Peptidomics.</a> Journal of Chromatography B. 803, 3-16 (2004).</li><li>Theodorsson, E., Stenfors, C., Mathe, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave+irradiation+increases+recovery+of+neuropeptides+from+brain+tissues.">Microwave irradiation increases recovery of neuropeptides from brain tissues.</a> Peptides. 11, 1191-1197 (1990).</li><li>Che, F. Y., Lim, J., Pan, H., Biswas, R., Fricker, L. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Intracellular+Peptides+from+Zebrafish+(Danio+rerio)+Brain.">Characterization of Intracellular Peptides from Zebrafish (Danio rerio) Brain.</a> Zebrafish. 16 (3), 240-251 (2019).</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=Analysis+of+mouse+brain+peptides+using+mass+spectrometry-based+peptidomics:+implications+for+novel+functions+ranging+from+non-classical+neuropeptides+to+microproteins.">Analysis of mouse brain peptides using mass spectrometry-based peptidomics: implications for novel functions ranging from non-classical neuropeptides to microproteins.</a> Molecular BioSystems. 6 (8), 1355-1365 (2010).</li><li>Gelman, J. S., Sironi, J., Castro, L. M., Ferro, E. S., 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=Peptidomic+analysis+of+human+cell+lines.">Peptidomic analysis of human cell lines.</a> Journal of Proteome Research. 10 (4), 1583-1592 (2011).</li><li>De Araujo, C. 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=Intracellular+peptides+in+cell+biology+and+pharmacology.">Intracellular peptides in cell biology and pharmacology.</a> Biomolecules. 9, 150 (2019).</li><li>Gewehr, M. C. F., Silverio, R., Rosa-Neto, J. C., Lira, F. S., Reckziegel, P., Ferro, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptides+from+natural+or+rationally+designed+sources+can+be+used+in+overweight,+obesity,+and+type+2+diabetes+therapies.">Peptides from natural or rationally designed sources can be used in overweight, obesity, and type 2 diabetes therapies.</a> Molecules. 25 (5), 1093 (2020).</li><li>Sakaya, G. R., 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=Peptidomic+profiling+of+cerebrospinal+fluid+from+patients+with+intracranial+saccular+aneurysms.">Peptidomic profiling of cerebrospinal fluid from patients with intracranial saccular aneurysms.</a> Journal of Proteomics. 240 (3), 104188 (2021).</li><li>Fricker, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+peptidomics:+General+considerations.">Quantitative peptidomics: General considerations.</a> Methods in Molecular Biology. 1719, 121-140 (2018).</li><li>Southey, B. R., 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=Comparing+label-free+quantitative+peptidomics+approaches+to+characterize+diurnal+variation+of+peptides+in+the+rat+suprachiasmatic+nucleus.">Comparing label-free quantitative peptidomics approaches to characterize diurnal variation of peptides in the rat suprachiasmatic nucleus.</a> Analytical Chemistry. 86 (1), 443-452 (2014).</li><li>Chen, X., Wei, S., Ji, Y., Guo, X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomics+using+SILAC:+Principles,+applications,+and+developments.">Quantitative proteomics using SILAC: Principles, applications, and developments.</a> Proteomics. 15 (18), 3175-3192 (2015).</li><li>Boonen, K., 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=Quantitative+peptidomics+with+isotopic+and+isobaric+tags.">Quantitative peptidomics with isotopic and isobaric tags.</a> Methods in Molecular Biology. 1719, 141-159 (2018).</li><li>Gewehr, M. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+peptide+stable+isotope+dimethyl+labeling+for+quantitative+proteomics.">Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.</a> Nature Protocols. 4 (4), 484-494 (2009).</li><li>Dasgupta, S., Castro, L. M., Tashima, A. K., Fricker, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+peptidomics+using+reductive+methylation+of+amines.">Quantitative peptidomics using reductive methylation of amines.</a> Methods in Molecular Biology. 1719, 161-174 (2018).</li><li>Tashima, A. K., Fricker, L. 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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...

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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 ...

Here, we describe an instructional method based on a fast heat inactivation of protease to preserve natural intracellular peptides, avoid degradation in cells and tissues, followed by relative quantitation of peptides using isotopic labeling. This labeling method has so much advantages. The reagents are commercially available, inexpensive, chemically stable, and allows the analysis of up to five samples in a single serum.Begin by cultivating human neuroblastoma cells in a 15 centimeter dish in DMEM containing 15%FBS and 1%penicillin streptomycin. Maintain the cells at 37 degrees Celsius under 5%carbon dioxide. Wash the cells twice with PBS, then add 10 milliliters of PBS, scrape the cells, and collect them into a 15 milliliter tube.Centrifugate the cells at 800 times G for five minutes and remove the supernatant. Resuspend the pellet in one milliliter of ultra purified deionized water at 80 degrees Celsius, then transfer the contents of the tube to a two milliliter microfuge tube. After heat inactivation of zebrafish, collect the whole brain in a two milliliter microfuge tube and freeze it at minus 80 degrees Celsius until analysis.Resuspend the tissue sample in one milliliter of ultra purified deionized water at 80 degrees Celsius and sonicate with a probe using 30 pulses of one second. Incubate the cellular lysate or homogenate tissue at 80 degrees Celsius for 20 minutes, then cool it on ice for 10 to 30 minutes. Add 10 microliters of one molar hydrochloric acid stock solution for each one milliliter of sample volume.Mix by vortexing for 20 seconds and incubate on ice for 15 minutes. Centrifugate the cellular lysate or the homogenate tissue at 12, 000 times G in four degrees Celsius for 15 minutes. Collect the supernatant in low binding protein microcentrifuge tubes and store it at minus 80 degrees Celsius.Clean the ultrafiltration devices with water and centrifuge at 2, 300 times G for three minutes, then discard the water from the ultrafiltration device. Pipette the supernatant into a pre-washed 10 kilodalton cutoff filter and spin in a refrigerated centrifuge at four degrees Celsius. Check the pH of the sample.Equilibrate the column with one milliliter of 100%acetonitrile, then wash it with one milliliter of 5%acetonitrile with 0.1%trifluoroacetic acid. Load the complete volume of the sample in the column and wash the column with one milliliter of 5%acetonitrile with 0.1%trifluoroacetic acid. Elute the peptides from the column with 1.8 milliliters of 1%acetonitrile with 0.15%trifluoroacetic acid into protein low binding microcentrifuge tubes.Dry the sample entirely in a vacuum centrifuge at 30 degrees Celsius and monitor the concentration time on display. Store the sample at minus 80 degrees Celsius. Resuspend the peptide samples in 100 to 200 microliters of ultra purified water and pipette 2.5 microliters of the standard peptide concentrations and samples onto the white 96-well plate.Add 25 microliters of 0.2 molar phosphate buffer and 12.5 microliters of fluorescamine. Homogenize gently for one minute on the orbital rotator. Next, add 110 microliters of water to stop the reaction.Adjust the settings on the spectrofluorometer, then read the plate. Add 1/10th volume of one molar trimethylammonium bromide to each peptide sample with up to 25 micrograms of the peptide. Ensure that the pH is between five and eight, adjusting with hydrochloric acid or sodium hydroxide, if needed.Add four microliters of non-deuterated, deuterated formaldehyde or carbon-13 deuterated formaldehyde. Mix for five seconds by vortexing. Add four microliters of sodium cyanoborohydride or deuterated sodium cyanoborohydride to the peptide sample.Then mix the sample for five seconds by vortexing. Incubate the peptide sample in a fume hood for two hours at room temperature, vortexing every 30 minutes. Repeat the addition of non-deuterated, deuterated, or carbon-13 deuterated formaldehyde and sodium cyanoborohydride or deuterated sodium cyanoborohydride, vortexing after each addition.Incubate the samples in the fume hood overnight at room temperature. Add 16 microliters of ammonium bicarbonate and mix by vortexing. Place the sample on ice.Add eight microliters of 5%formic acid and vortex for another five seconds. Combine the peptide samples, adjust the pH between two and four, then desalt the combined samples on reversed phase cleanup columns using acetonitrile and trifluoroacetic acid as previously described. Dry the sample entirely in a vacuum centrifuge at 30 degrees Celsius, then store it at minus 20 degrees Celsius.The labeled peptides are observed using mass spectra. Red arrows indicate the presence of peak pairs of different peptides labeled with two isotopic forms, L1 and L5, for comparison between two different samples S1 and S2 respectively. The mass spectrum of a peptide present in three different samples, S1, S2, and S3, labeled with L1, L3, and L5 tags respectively is shown here.A quadruplex labeling was performed. Control samples S1 and S3 were labeled with L1 and L3 respectively and compared with two experimental samples, S2 and S4 labeled with L2 and L4.No significant difference was observed. The isotopic labeling was used to show substrates in products in vitro for a given protease or peptidase.A peptide that does not change in the presence of the neurolysin enzyme is shown here. Peptides that disappear or reduce in the presence of the enzyme are substrates, while those that increase are considered products. The figure is an example of identifying a peptide sequence performed by a database search engine labeled with three different forms of tags.Before it's defined, ensure the sample is completely cool to avoid breaking the peptide bonds caused by hard acidification. In addition, the cleaning of ultrafiltration devices is essential. mass spectrometry is an excellent method to quantify peptides between different experimental conditions and for analysis studies by identifying peptidase substrates.

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Biochemistry

2.1K Views.  Sao Paulo State University (UNESP). Here, we describe an instructional method based on a fast heat inactivation of protease to preserve natural intracellular peptides, avoid degradation in cells and tissues, followed by relative quantitation of peptides using isotopic labeling. This labeling method has so much advantages. The reagents are commercially available, inexpensive, chemically stable, and allows the analysis of up to five samples in a single serum.Begin by cultivating human neuroblastoma cells in a 15 centimeter...