Portions of the work were supported by NIH Grants R01 DK122160, R01 HL139335, and U24 DK112349

All procedures described in the protocol related to animal or human samples/tissues were approved by and followed the institutional guidelines of the human and animal research ethics committee.
1. Sample homogenization/lysis
<ol>
	<li>Frozen tissue samples
	<ol>
		<li>Mince frozen tissue (~30 mg) on a glass microscope slide on dry ice using a prechilled razor blade and forceps. Transfer the minced tissue to a prechilled 5 mL round-bottom polystyrene tube containing 700 &#181;L of buffer A (s...

Resin-assisted capture has been utilized across a variety of sample types and biological systems for the investigation of oxidative modifications of cysteine residues25,29,30. This method allows for the evaluation of samples at multiple levels and readouts, including proteins and peptides using SDS-PAGE and western blot analysis, as well as individual cysteine sites using mass spectrometry. Regardless of the sample type or the f...

Under homeostatic conditions, cells generate reactive oxygen, nitrogen, or sulfur species that help to facilitate processes, such as metabolism and signaling1,2,3, extending to both prokaryotes and eukaryotes. Physiological levels of these reactive species are necessary for proper cellular function, also known as &#39;eustress&#39;1,4. In contrast, an increase in oxidants that leads to an imbalance between oxidants and antioxidants can cause oxidative stress, or &#39;distress&#39;1, which leads to cellular damage. Oxidants transduce signals to biological pathways by modifying different biomolecules, including protein, DNA, RNA, and lipids. In particular, cysteine residues of proteins are highly reactive sites prone to oxidation due to the thiol group on cysteine, which is reactive towards different types of oxidants5. This gives rise to a diverse range of reversible redox-based posttranslational modifications (PTMs) for cysteine, including nitrosylation (SNO), glutathionylation (SSG), sulfenylation (SOH), persulfidation (SSH), polysulfidation (SSnH), acylation, and disulfides. Irreversible forms of cysteine oxidation include sulfinylation (SO2H) and sulfonylation (SO3H).
Reversible oxidative modifications of cysteine residues may serve protective roles preventing further irreversible oxidation or serve as signaling molecules for downstream cellular pathways6,7. The reversibility of some thiol redox PTMs allows cysteine sites to function as &#34;redox switches&#34;8,9, wherein changes in the redox state of these sites alter protein function to regulate their role in transient processes. The modulatory effects of redox PTMs10 have been observed in many aspects of protein function11, including catalysis12, protein-protein interactions13, conformation change14, metal ion coordination15, or pharmacological inhibitor binding16. Additionally, redox PTMs are involved in cysteine sites of proteins that regulate pathways such as transcription17, translation18, or metabolism19. Given the impact that redox PTMs have on protein function and biological processes, it is important to quantify the extent of oxidation that a cysteine site undergoes in response to a perturbation of the redox state.
The identification of cysteine sites with altered redox states is focused on the comparison of the oxidation state at the site-specific level between normal and perturbed conditions. Fold change measurements are often utilized to determine what sites are significantly altered as this helps users interpret what cysteine sites may be physiologically significant to the study. Alternatively, stoichiometric measurements of reversible thiol oxidation across a specific sample type give a general picture of the physiological state with respect to cellular oxidation, an important measurement that is often overlooked and underutilized. Modification stoichiometry is based on quantifying the percentage of modified thiol as a ratio to total protein thiol (modified and unmodified)20,21. As a result, stoichiometric measurements offer a more precise measurement than fold change, especially when using mass spectrometry. The significance of the increase in oxidation can be more readily ascertained by using stoichiometry to determine the PTM occupancy of a particular cysteine site. For example, a 3-fold increase in thiol oxidation could result from a transition of as little as 1% to 3% or as big as 30% to 90%. A 3-fold increase in oxidation for a site that is only at 1% occupancy may have little impact on a protein&#39;s function; however, a 3-fold increase for a site with 30% occupancy at resting state may be more substantially affected. Stoichiometric measurements, when performed between total oxidized thiols and specific oxidative modifications, including protein glutathionylation (SSG) and nitrosylation (SNO), can reveal ratios and quantitative information with respect to specific modification types.
Because reversible thiol oxidation is typically a low-abundance posttranslational modification, multiple approaches have been developed for the enrichment of proteins containing these modifications out of biological samples. An early approach devised by Jaffrey and others, named the biotin switch technique (BST)22, involves multiple steps wherein unmodified thiols are blocked through alkylation, reversibly modified thiols are reduced to nascent free thiols, nascent free thiols are labeled with biotin, and the labeled proteins are enriched by streptavidin affinity pulldown. This technique has been used to profile SNO and SSG in many studies and can be adapted to probe for other forms of reversible thiol oxidation23,24. While BST has been utilized to probe for different forms of reversible thiol oxidation, one concern with this approach is that enrichment is impacted by the non-specific binding of unbiotinylated proteins to streptavidin. An alternate approach developed in our&#160;laboratory, named resin-assisted capture (RAC)25,26 (Figure 1), circumvents the issue of enrichment of thiol groups via the biotin-streptavidin system.
Following the reduction of reversibly oxidized thiols, proteins with nascent free thiols are enriched by the thiol-affinity&#160;resin, which covalently captures free thiol groups, allowing for more specific enrichment of cysteine-containing proteins than BST. Coupling RAC with the multiplexing power of the recent advances in isobaric labeling and mass spectrometry creates a robust and sensitive workflow for the enrichment, identification, and quantification of reversibly oxidized cysteine residues at the proteome-wide level. Recent advances in mass spectrometry have enabled much deeper profiling of the thiol redox proteome, increasing the understanding of both the cause and effect of protein thiol oxidation27. The information gained from site-specific quantitative data allows for further studies of the mechanistic impacts and downstream effects of reversible oxidative modifications28. Utilizing this workflow has provided insight into the physiological impacts of reversible cysteine oxidation with respect to normal physiological events such as aging, wherein levels of SSG differed with respect to age. The aging effects on SSG were partially reversed using SS-31 (elamipretide), a novel peptide that enhances mitochondrial function and reduces SSG levels in aged mice, causing them to have an SSG profile more similar to young mice29.
Pathophysiological conditions attributed to nanoparticle exposure have been shown to involve SSG in a mouse macrophage model. Using RAC coupled with mass spectrometry, the authors showed that SSG levels were directly correlated to the degree of oxidative stress and impairment of macrophage phagocytic function. The data also revealed pathway-specific differences in response to different engineered nanomaterials that induce different degrees of oxidative stress30. The method has also proven its utility in prokaryotic species, where it was applied to study the effects of diurnal cycles in photosynthetic cyanobacteria with respect to thiol oxidation. Broad changes in thiol oxidation across several key biological processes were observed, including electron transport, carbon fixation, and glycolysis. Furthermore, through orthogonal validation, several key functional sites were confirmed to be modified, suggesting regulatory roles of these oxidative modifications6.
Herein, we describe the details of a standardized workflow (Figure 1), demonstrating the utility of the RAC approach for the enrichment of total oxidized cysteine thiols of proteins and their subsequent labeling and stoichiometric quantification. This workflow has been implemented in studies of the redox state in different sample types, including cell cultures27,30 and whole tissues (e.g., skeletal muscle, heart, lung)29,31,32,33. While not included here, the RAC protocol is also easily adapted for the investigation of specific forms of reversible redox modifications, including SSG, SNO, and S-acylation, as previously mentioned25,29,34.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-(Pyridyldithio)ethylamine hydrochloride</td>
			<td>Med Chem Express</td>
			<td>HY-101794</td>
			<td>Reagent for in-house resin synthesis</td>
		</tr>
		<tr>
			<td>2.0 mL LoBind centrifuge tubes</td>
			<td>Eppendorf</td>
			<td>22431048</td>
			<td></td>
		</tr>
		<tr>
			<td>5.0 mL LoBind centrifuge tubes</td>
			<td>Eppendorf</td>
			<td>30108310</td>
			<td></td>
		</tr>
		<tr>
			<td>5.0 mL round bottom tubes</td>
			<td>Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A949-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr>
			<td>Activated Thiol&#8211;Sepharose&#160;4B</td>
			<td>Sigma Aldrich</td>
			<td>T8512</td>
			<td>Potential replacement for thiol-affinity resin</td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5 mL centrifugal filter</td>
			<td>Millipore Sigma</td>
			<td>UFC5010BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>09830</td>
			<td></td>
		</tr>
		<tr>
			<td>Bicinchonicic acid (BCA)</td>
			<td>Thermo Scientific</td>
			<td>23227</td>
			<td>Protein Assay Reagent</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Thermo Scientific</td>
			<td>20291</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>BioSpec Products</td>
			<td>985370</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetimide (IAA)</td>
			<td>Sigma Aldrich</td>
			<td>I1149</td>
			<td></td>
		</tr>
		<tr>
			<td>N-ethylmaleimide</td>
			<td>Sigma Aldrich</td>
			<td>4259</td>
			<td></td>
		</tr>
		<tr>
			<td>NHS-Activated Sepharose 4 Fast Flow</td>
			<td>Cytiva</td>
			<td>17-0906-01</td>
			<td>Reagent for in-house resin synthesis</td>
		</tr>
		<tr>
			<td>QIAvac 24 Plus vacuum manifold</td>
			<td>Qiagen</td>
			<td>19413</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma Aldrich</td>
			<td>L6026</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Branson</td>
			<td>1510R-MT</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin columns</td>
			<td>Thermo Scientific</td>
			<td>69705</td>
			<td></td>
		</tr>
		<tr>
			<td>Strata C18-E reverse phase columns</td>
			<td>Phenomenex</td>
			<td>8B-S001-DAK</td>
			<td>Peptide desalting</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5355</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiopropyl Sepharose 6B</td>
			<td>GE Healthcare</td>
			<td>17-0420-01</td>
			<td>Thiol-affinity resin; *Production of Thiopropyl Sepharose 6B resin has been discontinued by the manufacturer (see protocol for details).</td>
		</tr>
		<tr>
			<td>TMT isobaric labels (16 plex)</td>
			<td>Thermo Scientific</td>
			<td>A44522</td>
			<td>Peptide labeling reagent; available in multiple formats</td>
		</tr>
		<tr>
			<td>Triethylammonium bicarbonate buffer (TEAB)</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>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V5820</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma Aldrich</td>
			<td>U5378</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacufuge Plus speedvac</td>
			<td>Eppendorf</td>
			<td>22820001</td>
			<td>vacuum concentrator</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
	</tbody>
</table>

resin-assisted capture coupled with isobaric tandem mass tag labeling for multiplexed quantification of protein thiol oxidation

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. 
The workflow is adaptable across many sample types, including cultured cells (e.g., mammalian, prokaryotic) and whole tissues (e.g., heart, lung, muscle), which are initially lysed/homogenized and with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol-affinity resin, which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, the labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves the understanding of the role of redox-dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

Completion of the protocol will result in highly specific enrichment of formerly oxidized cysteine-containing peptides, often with &#62;95% specificity27,35,36. However, several key steps of the protocol require special attention, e.g., the initial blocking of free thiols prior to sample lysis/homogenization, which prohibits artificial oxidation and non-specific enrichment of artificially oxidized thiols25</sup...

Watch this Scientific Journal Video about Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation at JoVE.com

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Protein thiol oxidation has significant implications under normal physiological and pathophysiological conditions. We describe the details of a quantitative redox proteomics method, which utilizes resin-assisted capture, isobaric labeling, and mass spectrometry, enabling site-specific identification and quantification of reversibly oxidized cysteine residues of proteins.

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed ...

This protocol describes a method that enables site-specific identification of oxidized cystine residues of proteins. The method allows quantitative and site-specific data of oxidized cystine residues to be generated from various sample types. To begin the assisted capture procedure, wash the precipitated protein pellets twice with acetone by centrifuging at 20, 500 G, in four degrees Celsius for 10 minutes.Then decant the acetone and remove the remaining acetone with a micro pipette. Using a glass serological pipette add three milliliters of fresh ice cold acetone to the tube and mix well by inverting the tube several times. After the second wash, allow the pellets to air dry for one to two minutes without letting them over dry.Add one milliliter of buffer B to the dried protein pellet. To solubilize the protein, sonicate the pellet repeatedly in a bath sonicator with an output of 250 watts for 15 to 30 seconds at a time, along with brief vortexing. Perform the Bicinchoninic acid or BCA assay to measure the protein concentration.To standardize the protein concentrations, transfer 500 micrograms of protein sample to a 0.5 milliliter, 10 kilodaltons centrifugal filter, and volume up the sample to 500 microliters with resuspension buffer. Centrifuge the protein buffer mixture at 14, 000 G in room temperature, until the volume in the centrifugal filter is less than 100 microliters. Collect the sample by inverting the filter in a collection tube.Centrifuge the sample at 1000 G for two minutes and add buffer C to get a final volume of 500 microliters. To reduce the protein thiols, add 20 microliters of 500 millimolar dithiothreitol or DTT, to the sample to get a final concentration of 20 millimolar, and incubate the samples for 30 minutes at 37 degrees Celsius with shaking at 850 RPM. After reduction, centrifuge to the samples into 0.5 milliliter 10 kilodalton centrifugal filters at 14, 000 G at room temperature, until the volume in the centrifugal filter is less than 100 microliters.Add buffer D to make up the volume to 500 microliters and repeat centrifugation. After the fourth centrifugation, collect the samples by inverting the filter in a collection tube to centrifuge at 1000 G for two minutes. Then measure the protein concentration using the BCA assay.Next, place the spin columns on a vacuum manifold, and using a micro pipette transfer 500 microliters of the freshly prepared thiol affinity resin slurry to each column. Apply vacuum for removal of water. Repeat the procedure once to obtain 30 milligrams of resin per column.Wash the resin five times with 500 microliters of ultrapure water, by applying a vacuum to remove the water and then five times with 500 microliters of buffer E.In a new tube, add buffer C to 150 micrograms of the reduced protein sample until the final volume is 120 microliters. Transfer the protein solution to a plugged spin column containing the resin. Place the cap on the column and incubate for two hours at room temperature with shaking at 850 rpm.Wash the resin as described in the manuscript. Replace the plug. To perform on-resin tryptic digestion, add 120 microliters of freshly prepared trypsin solution to the samples, and incubate the column overnight at 37 degrees Celsius, with shaking at 850 RPM.On the next day, wash the resin multiple times as described in the manuscript and replace the plug. Using a micro pipette, add 40 microliters of 100 millimolar Triethylammonium bicarbonate buffer to the washed resin. Then add 70 microliters of the dissolved tandem mass tag or TMT labeling reagent, and incubate for one hour at room temperature with shaking at 850 RPM.Store any remaining TMT reagent at minus 80 degrees Celsius. Wash the resin and replace the plug. Elute the TMT labeled peptides by adding 100 microliters of 100 millimolar ammonium bicarbonate buffer at pH 8.0, containing 20 millimolar DTT, to the column, and incubating the column on a thermal mixer at room temperature for 30 minutes at 850 RPM.After incubation, perform elution by placing the column on a vacuum manifold, applying the vacuum and eluting the samples into a five milliliter micro centrifuge tube. Repeat the step once, then add 100 microliters of 80%acetonitrile with 0.1%trifluoroacetic acid to the column. After incubating the column for 10 minutes at room temperature, elute the sample in the same five milliliter centrifuge tube.Place the eluted samples in a vacuum concentrator until dry. Then store the dry peptides at minus 80 degrees Celsius until further use. The qualitative analysis of peptide samples from RAW 264.7 cells was carried out with SDS-PAGE, wherein samples were compared by determining different levels of oxidation due to treatments.The separated proteins were subsequently probed by western blot to further investigate the oxidation levels of individual proteins. Reported ion intensities of cystine containing peptides were analyzed by liquid chromatography tandem mass spectrometry. The thiol oxidation stoichiometry of iTRAQ, labeled enriched and oxidized peptides at individual cystine site levels were quantified.Carefully remove acetone without disturbing the protein pellet. Ensure that the protein pellet is completely solubilized and quantitated precisely, and that resin is accurately allocated to spin columns. Be sure to resuspend resin during the wash steps.Further evaluation of enriched proteins and peptides may be performed by SDS-PAGE and staining, western blotting and mass spectrometry.

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Biochemistry

1.6K 조회수.  Pacific Northwest National Laboratory. 이 프로토콜은 단백질의 산화된 시스틴 잔기의 부위 특이적 식별을 가능하게 하는 방법을 설명합니다. 이 방법을 사용하면 산화된 시스틴 잔기의 정량적 및 부위별 데이터를 다양한 샘플 유형에서 생성할 수 있습니다. 보조 포획 절차를 시작하려면 침전 된 단백질 펠릿을 섭씨 4도에서 섭씨 4도에서 20, 500G에서 10 분 동안 원심 분리하여 아세톤으로 두 번 세척하십시오.?...