This work was supported by American Cancer Society Institutional Research Grant IRG-14-195-01 (M. Sharon Stack is Principal Investigator; X.L. is a subrecipient investigator). This publication was made possible with partial support from Grant Numbers KL2 TR002530 and UL1 TR002529 (A. Shekhar, PI) from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award. X. L. is a recipient of Indiana CTSI KL2 Young Investigator Award. S. F. is supported by Walther Cancer Foundation Advancing Basic Cancer grants. X. W. is supported by the National Natural Science Foundation of China General Program (Grant No. 817773047).

1. Nitration of Angiotensin II
<ol>
	<li>To generate nitrated peptide, dilute 10 &#956;L of Angiotensin II (DRVYIHPF) parent solution (2 mM in water) in 390 &#956;L PBS solution (10 mM NaH2PO4, 150 mM NaCl, pH 7.4) at the final concentration of 50 &#956;M.</li>
	<li>Add 10 &#956;L of peroxynitrite (200 mM in 4.7% NaOH) to the Angiotensin II solution to make the final concentration of peroxynitrite 5 mM. 
	NOTE: Peroxynitrite is unstable and easy to resolve when it is in acid form, so parent s...

<ol>
	<li>Radi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide,+oxidants,+and+protein+tyrosine+nitration.">Nitric oxide, oxidants, and protein tyrosine nitration.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 4003-4008 (2004).</li><li>Radi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+tyrosine+nitration:+biochemical+mechanisms+and+structural+basis+of+functional+effects.">Protein tyrosine nitration: biochemical mechanisms and structural basis of functional effects.</a> Accounts of Chemical Research. 46, 550-559 (2013).</li><li>Zhan, X., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-induced+fragmentation+of+leucine+enkephalin,+nitro-Tyr-leucine+enkaphalin,+and+d5-Phe-nitro-Tyr-leucine+enkephalin.">MALDI-induced fragmentation of leucine enkephalin, nitro-Tyr-leucine enkaphalin, and d5-Phe-nitro-Tyr-leucine enkephalin.</a> Int. J Mass Spectrometry. 287, 77-86 (2009).</li><li>Batthy&#225;ny, 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=Tyrosine-Nitrated+Proteins:+Proteomic+and+Bioanalytical+Aspects.">Tyrosine-Nitrated Proteins: Proteomic and Bioanalytical Aspects.</a> Antioxidants &amp; Redox Signaling. 26, 313-328 (2017).</li><li>Feeney, M. B., Sch&#246;neich, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+approaches+to+analyze+protein+tyrosine+nitration.">Proteomic approaches to analyze protein tyrosine nitration.</a> Antioxidants &amp; Redox Signaling. 19, 1247-1256 (2013).</li><li>Zhan, X., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitroproteins+from+a+human+pituitary+adenoma+tissue+discovered+with+a+nitrotyrosine+affinity+column+and+tandem+mass+spectrometry.">Nitroproteins from a human pituitary adenoma tissue discovered with a nitrotyrosine affinity column and tandem mass spectrometry.</a> Analytical Biochemistry. 354, 279-289 (2006).</li><li>Zhan, X., Wang, X., Desiderio, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+analysis+of+nitrotyrosine-containing+proteins.">Mass spectrometry analysis of nitrotyrosine-containing proteins.</a> Mass Spectrometry Reviews. 34, 423-448 (2015).</li><li>Abello, N., Barroso, B., Kerstjens, H. A. M., Postma, D. 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The authors have nothing to disclose.

The protocol here describes the nitropeptide enrichment and profiling. Using Angiotensin II as the model peptide, we illustrated the procedure shown in Figure 1. After obtaining the nitro-Angiotensin II, the primary amine on the peptide should be blocked to avoid the further amine conjugation, which is one of the most critical steps within the protocol. In the current protocol, dimethyl labeling is used to block the primary amines for two reasons: first, it enables the acquisition of quantit...

Nitration of tyrosine residues in proteins to form 3-nitrotyrosine regulates many biological processes. Due to the different chemical properties between tyrosine and 3-nitrotyrosine, a nitrated protein may have perturbed signaling activity1,2. Therefore, it is important to develop methods that can enrich and identify nitration sites on proteins efficiently. As 3-nitrotyrosine is a low abundance modification on proteins compared to other forms of PTM, such as phosphorylation and acetylation, it is challenging to identify endogenous nitration sites directly from cell lines or tissue samples. Nevertheless, the methodology of using mass spectrometry (MS) to characterize the fragmentation pattern of nitrotpeptide has been developed (for example, Zhan &#38; Desiderio3), which lays the foundation for new methods of nitroproteomics.&#160;
Currently, an enrichment step followed by MS is the most powerful strategy for nitropeptide profiling4,5. The enrichment methods can be classified into two classes. One class is based on antibodies that can recognize 3-nitrotyrosine specifically, whereas the other class is based on the chemical derivation that reduces a nitro group to an amine group4,5. For the antibody-based method, nitrotyrosine affinity column is used for the enrichment, from which the eluted material is further resolved and analyzed by high resolution MS6,7. For the chemical derivation-based method, the amine groups at the N-terminus of the peptide or lysine should be blocked in the first step either by acetylation, isobaric tags for relative and absolute quantitation (iTRAQ), or tandem mass tags (TMT) reagents. Next, a reducer is used to reduce nitrotyrosine to aminotyrosine followed by modifying the newly formed amine group, which includes biotin ligation, sulphydryl peptide conversion, or other types of tagging systems8,9,10,11. Most of the protocols established so far are based on in vitro over-nitrated proteins, instead of endogenously nitrated proteins.
In the present study, a modified procedure of the chemical derivation of nitrotyrosine is developed for the nitropeptide enrichment and identification, which shows enhanced sensitivity during MS detection and is suitable for quantification purpose. Our recent study employing this method in biological systems identified that nitration of lymphocyte-specific protein tyrosine kinase (LCK) at Tyr394 by RNS produced from myeloid-derived suppressor cells (MDSCs) plays an important role in the immunosuppression of the tumor microenvironment12. Therefore, our method of nitropeptide identification can be applied to complex biological samples as well. Here, we describe our protocol by using the model peptide Angiotensin II, of which the fragmentation pattern is known and widely used in nitroproteomic studies8,9,10,11, as an example.

<table border="0" cellpadding="0" cellspacing="0" style="width:756px;" width="756">
	<tbody>
		<tr height="20">
			<td height="20">Acclaim pepmap 100 C18 column</td>
			<td style="width:197px;">Thermo-Fisher</td>
			<td style="width:119px;">164534</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="20">
			<td height="20">1 M TEAB solution</td>
			<td>Sigma-Aldrich</td>
			<td>T7408</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20">50% hydroxylamine</td>
			<td>Thermo-Fisher</td>
			<td>90115</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20">Acetonitrile</td>
			<td>Thermo-Fisher</td>
			<td>A955</td>
			<td>MS Grade</td>
		</tr>
		<tr height="20">
			<td height="20">dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>43819</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20">formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td>Molecular Biology Grade</td>
		</tr>
		<tr height="22">
			<td height="22">formaldehyde-D2</td>
			<td>Toronto Research Chemicals</td>
			<td>F691353</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22">formic acid</td>
			<td>Sigma-Aldrich</td>
			<td>695076</td>
			<td>ACS reagent</td>
		</tr>
		<tr height="22">
			<td height="22">Fusion Lumos mass spectrometer</td>
			<td>Thermo</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20">isoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I1149</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20">Methanol</td>
			<td>Thermo-Fisher</td>
			<td>A456</td>
			<td>MS Grade</td>
		</tr>
		<tr height="20">
			<td height="20">NHS-S-S-bition</td>
			<td>Thermo-Fisher</td>
			<td>21441</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20">Oasis HLB column (10 mg)</td>
			<td>Waters</td>
			<td>186000383</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20">peroxynitrite</td>
			<td>Merck-Millipore</td>
			<td>516620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20">sodium cyanoborohydrite</td>
			<td>Sigma-Aldrich</td>
			<td>42077</td>
			<td>PhamaGrade</td>
		</tr>
		<tr height="20">
			<td height="20">sodium dithionate</td>
			<td>Sigma-Aldrich</td>
			<td>157953</td>
			<td>Technical Grade</td>
		</tr>
		<tr height="20">
			<td height="20">Streptavidin Sepharose</td>
			<td>GE Healcare</td>
			<td>GE17-5113-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20">Ultimate 3000 nanoLC</td>
			<td>Thermo</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

nitropeptide profiling and identification illustrated by angiotensin ii

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

The flowchart for nitropeptide profiling in this manuscript is shown in Figure 1. Figure 2, 3, 4 and 5 show the mass spectra of Angiotensin II, nitro-Angiotensin II, dimethyl labeled nitro-Angiotensin II and dimethyl labeled amino Angiotensin II, respectively. The molecular weight of the compound can be reflected by the m/z values of the mono-isotope peak in each figure, indicating the chemical modification on A...

Watch this Scientific Journal Video about Nitropeptide Profiling and Identification Illustrated by Angiotensin II at JoVE.com

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

This message that can help answer key questions about how to enrich and identify nitropeptides by a chemical approach and the mass spectrometry. This technique has an enhanced sensitivity for detecting nitropeptides by mass spectrometry and that can be applied to both in vitro biochemical systems and endogenous biological tissues. For nitro-angiotensin II desalting, use a one milliliter pipet to precondition a reverse-phase solid-phase extraction or SPE column with the addition of 500 microliters of methanol and 500 microliters of water into the top of the column.When all of the water has run through the column, load 410 microliters of nitro-angiotensin II solution onto the column. Wash the column with 500 microliters of 5%methanol and water and 300 microliters of 80%methanol and water to elute the nitropeptide. Then dry the eluate by speed vacuum in the default setting at room temperature.For primary amine alkylation, reconstitute the powdered nitro-angiotensin II in 100 microliters of 100 millimolar Triethylammonium bicarbonate solution and add 400 microliters of 4%formaldehyde to the solution in a fume hood with brief mixing. Next, add four microliters of 0.6 molar sodium cyanoborohydride to the solution with shaking at 400 rotations per minute for one hour at room temperature. At the end of the incubation, quench the labeling reaction with 16 microliters of 1%ammonia solution for five minutes at room temperature followed by acidification with eight microliters of formic acid.Then desalt the solution in a new reverse-phase SPE column as just demonstrated. For nitrotyrosine to aminotyrosine reduction, reconstitute the dimethyl-labeled nitro-angiotensin II in 500 microliters of PBS and add 10 microliters of one molar sodium dithionate for a one hour incubation at room temperature. After the reduction reaction, the yellow solution will become clear.Desalt the reduced sample in a new reverse-phase SPE column as demonstrated. For biotinylation and enrichment, reconstitute the dimethyl-labeled aminoangiotensin II in 500 microliters of PBS followed by the addition of five microliters of 40 nanomolar NHS-S-S biotin dissolved in dimethyl sulfoxide for a two hour incubation at room temperature. At the end of the incubation, quench the reaction with one microliter of 5%hydroxylamine and add 500 microliters of fresh PBS to 100 microliters of Streptavidin Agarose beads for the equilibration by three separate centrifugations.After the last centrifugation, add 100 microliters of beads to the reaction system for a one hour incubation on a Rotary Shaker at room temperature. At the end of the incubation, wash the system four times with 500 microliters of fresh PBS per wash followed by the addition of 400 microliters of 10 millimolar dithiothreitol for a 45 minute incubation at 50 degrees Celsius. At the end of the incubation, spin down the column and transfer the supernatant into a new tube containing 20 microliters of 0.5 molar iodoacetamide for a 20 minute incubation in the dark.Then desalt the solution in a new reverse-phase SPE column as demonstrated and resolve the dry peptide in 20 microliters of 0.1%formic acid. For detection, load the product onto a liquid chromatographer with a tandem mass spectrometer. Separate the modified angiotensin II peptides by a 60 minute gradient elusion at a flow rate of 0.3 microliters per minute with the nano-high pressure liquid chromatography system that is directly interfaced with the high-resolution mass spectrometer.In the data-dependent acquisition mode, set a single full scan mass spectrum in the mass spectrometer, followed by 20 data-dependent tandem mass spectrometer scans at 28%normalized collision energy. Then open the mass spectra data and identify the peaks of the product in each step to confirm the chemical reaction has been successfully performed. Here, representative mass spectra of angiotensin II before and after each chemical modification as demonstrated can be observed.The molecular weight of the compound is indicated by the mass-to-charge ratio values of the mono isotope peak, indicating that the chemical modification on angiotensin II was successfully achieved for that step. Here, the final product as detected and characterized by liquid chromatography with tandem mass spectrometry is shown. Quantitative analysis of the nitropeptides by dimethyl-labeling allows calculation of the relative amounts of light and heavy via comparison of the intensity of the mono isotope peak in each group, allowing quantification of the enriched nitropeptides from different groups.Following the procedure, you can use a program search engine such as the SEQUEST max count of pFIND to identify the potential nitropeptides. After its development, this technique paved the way for the starting of the biological functions of specific nitration sites.

nitropeptide-profiling-identification-illustrated-angiotensin

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JoVE Journal

Biochemistry

5.6K Görüntüleme.  Westlake University. Kimyasal bir yaklaşım ve kütle spektrometresi ile azotların nasıl zenginleştirilip tanımlanabileceği ile ilgili anahtar soruların cevaplanabildiği bu mesaj. Bu teknik, kütle spektrometresi ile nitropeptidlerin saptanması nda gelişmiş bir duyarlılığa sahiptir ve hem in vitro biyokimyasal sistemlere hem de endojen biyolojik dokulara uygulanabilir. Nitro-anjiyotensin II tuzsuzlaştırma için, bir mililitrelik pipet kullanarak bir ters faz katı faz ekstraksiyonunu veya SPE kolo...