VV was supported by the intramural research grant (Forschungsf&#246;rderung) of the University Medical Center G&#246;ttingen and the Else Kr&#246;ner research program of the Else Kr&#246;ner-Fresenius-Stiftung.

CAUTION: The method uses hydrochloric acid (HCl, starting dilutions from ultrapure, concentration 1 M) and tetramethylammoniumhydroxide (TMAH, starting dilutions from ultrapure, concentration 25%). Both substances are strongly corrosive. Use skin and eye protection.
1. Preparing electrolytes
<ol>
	<li>Preparing HCl-electrolytes: background electrolyte (20 mM HCl), outlet electrolyte (5 mM HCl) and terminating electrolyte (0.05 mM HCl)
	<ol>
		<li>Prepare 20 mM HCl in a 100 mL flask: Pipette 2 mL of 1 M HCl into the flask, fill up to the mark with ultrapure water and shake gently.</li>
		<li>Prepare 5 mM HCl in a 100 mL flask: Pipette 500 &#181;L of 1 M HCl into the flask, fill up to the mark with ultrapure water and shake gently.</li>
		<li>Prepare 0.05 mM HCl in two steps: Pipette 1 mL of 20 mM HCl into a 100 mL flask, and then fill up to the mark with ultrapure water and shake gently. Subsequently, pipette 2.5 mL of the latter solution into a 15 mL conical tube (Table of Materials) and add 7.5 mL of ultrapure water, then shake gently.</li>
	</ol>
	</li>
	<li>Preparing leading electrolyte 12% TMAH in a 15 mL conical tube: Pipette 4.8 mL of 25% TMAH into the tube, add 5.2 mL of ultrapure water and shake gently. 
	NOTE: The 12% TMAH is used to purge and clean the capillary before each run and as a leading electrolyte in front of the injected sample).</li>
</ol>
2. Preparation and storage of standards and samples
<ol>
	<li>Standards
	<ol>
		<li>For Fe(II), weigh 35.61 mg of Fe(II)Cl2&#183;4H2O into a 100 mL flask and fill up to the 100 mL mark with ultrapure water for a 100 mg Fe(II)/L stock concentration. Shake gently until complete dissolution.</li>
		<li>For Fe(III), weigh 29.04 mg of Fe(III)Cl3 into a 100 mL flask and fill up to the 100 mL mark with ultrapure water for a 100 mg Fe(III)/L stock concentration. Shake gently until complete dissolution.</li>
		<li>Dilute each standard solution according to Table 1 to prepare the working standard solutions. 
		NOTE: After preparing the daily stock solutions from the 100 mg/L stock solution, the latter must be stored frozen. After preparing the daily standards according to Table 1, the 1 mg/L stock solution must be aliquoted into 1.5 mL volumes and stored frozen (best with no air left on top) in 1.5 mL tubes. For each new day, one daily stock cap is thawed for preparation of daily standards and withdrawn after use.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Starting concentration</td>
			<td>Pipetting volume</td>
			<td>Fill up with Milli-Q water</td>
			<td>Resulting concentration</td>
			<td>Final volume</td>
			<td>Use of solution</td>
		</tr>
		<tr>
			<td>100 mg/L</td>
			<td>50 &#181;L</td>
			<td>4950 &#181;L</td>
			<td>1 mg/L</td>
			<td>50 mL</td>
			<td>Daily stock solution</td>
		</tr>
		<tr>
			<td rowspan="2">1 mg/L</td>
			<td rowspan="2">200 &#181;L</td>
			<td rowspan="2">1800 &#181;L</td>
			<td rowspan="2">100 &#181;g/L</td>
			<td rowspan="2">2 mL</td>
			<td>Standard</td>
		</tr>
		<tr>
			<td>100 &#181;g/L</td>
		</tr>
		<tr>
			<td rowspan="2">1 mg/L</td>
			<td rowspan="2">100 &#181;L</td>
			<td rowspan="2">1900 &#181;L</td>
			<td rowspan="2">50 &#181;g/L</td>
			<td rowspan="2">2 mL</td>
			<td>Standard</td>
		</tr>
		<tr>
			<td>50 &#181;g/L</td>
		</tr>
		<tr>
			<td rowspan="2">1 mg/L</td>
			<td rowspan="2">50 &#181;L</td>
			<td rowspan="2">1950 &#181;L</td>
			<td rowspan="2">25 &#181;g/L</td>
			<td rowspan="2">2 mL</td>
			<td>Standard</td>
		</tr>
		<tr>
			<td>25 &#181;g/L</td>
		</tr>
		<tr>
			<td rowspan="2">1 mg/L</td>
			<td rowspan="2">25 &#181;L</td>
			<td rowspan="2">1975&#181;L</td>
			<td rowspan="2">12.5&#181;g/L</td>
			<td rowspan="2">2 mL</td>
			<td>Standard</td>
		</tr>
		<tr>
			<td>12.5 &#181;g/l</td>
		</tr>
		<tr>
			<td rowspan="2">1 mg/L</td>
			<td rowspan="2">20 &#181;L</td>
			<td rowspan="2">1980&#181;L</td>
			<td rowspan="2">10 &#181;g/L</td>
			<td rowspan="2">2 mL</td>
			<td>Standard</td>
		</tr>
		<tr>
			<td>10 &#181;g/L</td>
		</tr>
		<tr>
			<td></td>
			<td>0</td>
			<td>2000 &#181;L</td>
			<td>0 &#181;g/L</td>
			<td>2 mL</td>
			<td>Blank</td>
		</tr>
	</tbody>
</table>
Table 1: Pipetting scheme for preparing the standards.
<ol start="2">
	<li>SH-SY5Y cell lysate 
	NOTE: The cell lysate (SH-SY5Y) served as Fe(II)/(III)-relevant bio-matrix to show the performance and reliability of the method.
	<ol>
		<li>Use lysate from previously running experiments16. Follow this cell lysate preparation avoiding pH changes or chemicals that might affect redox balance. Use a modified radioimmunoprecipitation assay (RIPA) lysis buffer (PBS pH 7.4, 0.5% sodium deoxycholate, 1% NP-40), avoiding metal chelators (such as EDTA), reducing agents (such as DTT, 2-Mercaptoethanol) and anionic surfactant detergents and metal complexing agents (such as SDS) for minimizing post-collection alterations of the Fe(II)/Fe(III) ratio.</li>
		<li>Work under a N2-atmosphere inhibited oxidation by O2 from ambient air and work on ice to minimize any autoxidation until the lysate was stored as soon as possible at -80 &#176;C under the nitrogen atmosphere.</li>
	</ol>
	</li>
</ol>
3. Setting up instruments for hyphenation of CE to ICP-MS
<ol>
	<li>Set up the capillary electrophoresis instrument. 
	NOTE: For this section, the reader is mainly referred to the manual of the respective instrument available in the laboratory.
	<ol>
		<li>Install a capillary with a suitable length to reach from the inlet vial of the CE instrument to the nebulizer of ICP-MS. Install the capillary only at the inlet side and lead it outside the instrument toward CE-ICP-MS interface. 
		NOTE: For hyphenating CE to ICP-MS, in this protocol a 90 cm fused silica capillary (ID 50 &#181;m) was installed according to the general instrumental setup description. Typically, capillary sizes of 70&#8722;100 cm will be needed, depending on the position of the instruments in the laboratory.</li>
		<li>De-activate the outlet lift of the CE-instrument in the software for smooth operation as it is not in use when the capillary is directed outside to the CE-ICP-MS interface.</li>
		<li>Install a trigger cable from CE-instrument trigger-OUT to trigger-IN of the ICP-MS instrument.</li>
		<li>Select positions for all necessary solutions (20 mM HCl, 0.05 mM HCl, 12% TMAH), standards and samples in the sample &#38; solutions rotor of the instrument and define their positions in the instrument software as usual (refer to the manual of the instrument).</li>
		<li>Select rotor and capillary temperature to be identical at 20 &#176;C, as being identical to the controlled laboratory temperature. 
		NOTE: No temperature gradient to the capillary parts inside and outside the CE instrument occurs.</li>
	</ol>
	</li>
	<li>Set up the ICP-MS instrument
	<ol>
		<li>Optimize the ICP-MS instrument according to the daily instrumental standard setup and operation procedures. Use the manufacturer&#8217;s protocol.</li>
		<li>Use dynamic reaction cell (DRC) technology with NH3 as the DRC gas, with 0.6 mL/min NH3&#8211;flowrate and RPq value = 0.45. 
		NOTE: For iron speciation, a method is programmed with 56Fe, being the most abundant Fe isotope (91.754% relative abundance), however, being severely interfered from the [40Ar16O]+ cluster. A quadrupole based ICP-MS in standard mode is practically blind and the detector in overflow at this isotope. With the settings above (see step 3.2.2), low baselines and high sensitivity are achieved (for regular total iron determination LOD in the low ng/L range is achieved).</li>
		<li>Choose a dwell time setting per isotope at 50 ms for monitoring even sharp and short appearing peaks during CE-separation.</li>
		<li>Program the ICP-MS method to be trigger-started by the CE-instrument.</li>
	</ol>
	</li>
	<li>Set up the CE-ICP-MS interface 
	NOTE: There are mainly two options for connecting the CE-capillary to the ICP-MS. Follow the provided descriptions about setup if a commercial interface is used. This protocol uses a simple, homemade interface based on a previous publication after modifications20. Key issues are an efficient nebulization with possibly less dilution of the capillary efflux aside from adoption of the overall flowrate to nebulizer for best nebulization. Also, the minimization of a suction flow through separating capillary caused by the self-aspiration from nebulizer, and the electrical connection of the grounded outlet electrode to capillary end are mandatory.
	<ol>
		<li>Nebulizer selection
		<ol>
			<li>Use a concentric nebulizer with low self-aspirating volume (e.g., 100 &#181;L/min) that fits into a low-volume spray chamber. 
			NOTE: The low uptake will cause only moderate dilution of capillary efflux paralleled from the still optimized nebulization. Electrical connection of the outlet electrode is accomplished by an electrolyte flow around the outlet electrode and around the capillary end.</li>
			<li>Use the self-aspiration of the nebulizer to minimize the suction through the separating capillary and for adoption of the flow rate to the optimal value needed by the nebulizer.</li>
			<li>Prepare the following parts from Table 2 to mount this home-made interface.</li>
		</ol>
		</li>
		<li>Setup of the simple interface 
		NOTE: Use Figure 1 to follow the description of part mounting for the simple interface. The numbers in Figure 1 and in the following text refer to the numbers in Table 2.
		<ol>
			<li>Start mounting the interface by connecting the two 3-way female Luer connectors (No. 3) with a male cone Luer connector (No. 4). Connect the left end of the lower 3-way Luer bar to the male connector and that to the middle connection of the upper 3-way Luer.</li>
			<li>Put a 1 cm tube (No. 1) over the Pt-wire (No. 7) and a 1 cm silicone tube (No. 5) over the latter one and the nozzle of a male Luer connector (No. 2). Fix the assembly to the middle connection of the lower 3-way Luer connector (No. 3) by Luer-typical screw rotation.</li>
			<li>Push a 1 cm tube (No.1) over the outlet end of the CE capillary and position it about 8-9 cm from the end.</li>
			<li>Put a 1 cm silicone tube (No. 5) over the latter one and the nozzle of a male Luer connector (No. 2).</li>
			<li>Put the whole assembly from the left through the bar of the upper 3-way-T connector and fix the male Luer connector and left end of the female 3-way Luer connector (No. 3) by Luer-typical screw rotation.</li>
			<li>Fix the 25 cm silicone tube (No. 5) at the nozzle of a male Luer connector (No. 2) and fix the whole assembly at the lower (right) end of the bar from the lower 3-way Luer connector (No.3) by Luer-typical screw rotation.</li>
			<li>Take the 1 cm silicone tube (No.6) and push it 5 mm over the end of the nebulizer tightly while the second male Luer cone connector (No. 4) is plugged tightly into the protruding part of the silicone tube.</li>
			<li>Move the above mounted interface part subsequently with the protruding CE capillary to the male cone at the nebulizer. Insert the CE capillary carefully through the male cone and further through the wider part of the nebulizer capillary until the latter gets narrow. Given that the protruding length of the capillary was chosen suitably, the upper female 3-way Luer connector now also fits tightly to the male cone.</li>
			<li>Correct the length of the protruding capillary length if necessary, by moving the capillary (forward/backward) at the tube (No. 1) where entering the self-made interface. 
			NOTE: The optimal position of the CE capillary at the beginning of the nebulizer capillary is not too critical. However, do not push the CE capillary too close to the narrow part of nebulizer capillary. This could hinder or block the outlet electrolyte flow. Also, this would interrupt the electrical connection to the outlet electrode and would increase the suction through CE capillary, resulting in disturbed separation. In turn, do not keep the CE capillary end too far away from nebulizer capillary since then sharp separated peaks would be broadened and resolution will be lost.</li>
			<li>Use a lens to identify the best position. 
			NOTE: Figure 1 (bottom right) shows the optimal position of CE-capillary inside the nebulizer.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61055/61055fig1v2.jpg" /> 
Figure 1: Schematic and mounting of the CE-ICP-MS interface.&#160;The schematic identifies the single parts for stepwise mounting of the simple and cheap CE-ICP-MS interface. The window shows a photo of optimal positioning of the CE-capillary in the nebulizer. <a href="https://www.jove.com/files/ftp_upload/61055/61055fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>No.</td>
			<td>Part</td>
			<td>used for</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Tubing (green-orange color code), 2 x ca. 1 cm</td>
			<td>fixing CE capillary and outlet electrode at Luer-parts and keeping tight</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Luer, male, 3 x, suitable for 1.6 mm ID silicone tubing</td>
			<td>connection of silicone tube to outlet electrolyte and as aid for fixing CE capillary and Pt-wire electrode</td>
		</tr>
		<tr>
			<td>3</td>
			<td>3-way-Luer, female, 2 x</td>
			<td>T-pieces to connect electrode, capillary and aspirated outlet flow</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Luer cone, male, 2 x</td>
			<td>connecting female Luers to each other and to nebulizer</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Silicone tube, 1.6 mm ID, 0.8 mm wall, 2 x 1 cm, 1x ca. 25 cm</td>
			<td>a)&#160;&#160;&#160;&#160;&#160; 1cm; tighting CE capillary to interface, 
			b)&#160;&#160;&#160;&#160;&#160; 1cm; tighting Pt-wire to interface 
			c)&#160;&#160;&#160;&#160;&#160; 25 cm; connection from outlet electrolyte flask to interface</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Silicone tube, 3 mm ID, 1.2 mm wall, ca.1 cm</td>
			<td>tighting Luer cone to nebulizer</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Platinum wire</td>
			<td>Outlet electrode</td>
		</tr>
	</tbody>
</table>
Table 2: Parts for building the simple, self-made CE-ICP-MS interface. The numbers refer also to Figure 1 and description in the text.
4. Preparation for measurement
NOTE: Before measurement, the capillary should be flushed with strong alkaline solution (here: 12% TMAH) for cleaning and then filled with background electrolyte. For improved separation a stacking buffer sandwich is built around the sample based on conductivity and pH gradients. Table 3 summarizes the consecutive preparation steps of the capillary, which are processed automatically by the instrument according to programmed method:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step-No</td>
			<td>Step</td>
			<td>chemical</td>
			<td>condition</td>
		</tr>
		<tr>
			<td>&#160;</td>
			<td>Preparation of CE column</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prep 1</td>
			<td>Capillary cleaning</td>
			<td>12 % TMAH</td>
			<td>4 bar, 1 min.</td>
		</tr>
		<tr>
			<td>Prep 2</td>
			<td>Capillary purging with background electrolyte</td>
			<td>20 mM HCl</td>
			<td>4 bar, 1 min.</td>
		</tr>
		<tr>
			<td>Prep 3</td>
			<td>Stacking: leading electrolyte</td>
			<td>12 % TMAH</td>
			<td>150 mbar, 3 s</td>
		</tr>
		<tr>
			<td>Prep 4</td>
			<td>Injection</td>
			<td>Sample</td>
			<td>150 mbar, 3 s</td>
		</tr>
		<tr>
			<td>Prep 5</td>
			<td>Stacking: terminating electrolyte</td>
			<td>0.05 mM HCl</td>
			<td>150 mbar, 3 s</td>
		</tr>
	</tbody>
</table>
Table 3: Capillary preparation steps before measurement. These steps are programmed with the CE-system software in the CE-method and include pressurized sample injection and the build-up of a &#34;stacking sandwich&#34; around the sample.
<ol>
	<li>Program a CE method, which is executing consecutively the steps given in Table 3.</li>
	<li>Define a sample table and sequence in CE software and copy this sequence also into ICP-MS software.</li>
</ol>
5. Measurement and data evaluation
<ol>
	<li>Start the method at CE instrument. After the programmed preparation and filling of the capillary, measurement starts automatically as soon as the inlet vial, containing 20 mM HCl, is in position at capillary inlet. The &#8220;Start-trigger&#8221; is sent to the ICP-MS, which starts the on-line monitoring of Fe-isotopes. 
	NOTE: The separation uses a voltage of +25 kV. The extended length of the capillary, necessary to connect the CE-instrument to ICP-MS, causes separation time to be unnecessarily increased. Therefore, the separation is supported by a low pressure of 250 mbar at inlet. The self-aspirated electrolyte at outlet is 5 mM HCl. The total analysis lasts 3 minutes for samples with moderate conductivity. In the signal window of the ICP-MS software the electropherogram can be observed during the run. At the end of each sample, two data files are automatically generated, one only accessible from instrument software from internal data bank, a second one in the export folder as &#8220;.xl&#8221; or &#8220;.txt&#8221; format, accessible by import function from regular chromatography software.</li>
	<li>Refer to the software manual of the ICP-MS instrument for exporting the files into chromatography software.</li>
</ol>

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Injection of Manganese in Rats.</a> Plos One. 10 (9), (2015).</li><li>Venkataramani, V., 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=Manganese+causes+neurotoxic+iron+accumulation+via+translational+repression+of+Amyloid+Precursor+Protein+(APP)+and+H-Ferritin.">Manganese causes neurotoxic iron accumulation via translational repression of Amyloid Precursor Protein (APP) and H-Ferritin.</a> Journal of Neurochemistry. 147 (6), 831-848 (2018).</li><li>Willkommen, D., Lucio, M., Schmitt-Kopplin, P., Gazzaz, M., Schroeter, M., Sigaroudi, A., Michalke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+fractionation+in+a+case-control+study+concerning+Parkinson's+disease:+Cu-amino+acids+discriminate+CSF+of+PD+from+controls.">Species fractionation in a case-control study concerning Parkinson's disease: Cu-amino acids discriminate CSF of PD from controls.</a> Journal of Trace Elements in Medicine and Biology. 49, 164-170 (2018).</li><li>Thibault, P., Dovichi, N. J., Camilleri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+instrumentation+and+detection+systems+including+mass+spectrometry.">General instrumentation and detection systems including mass spectrometry.</a> Capillary Electrophoresis - Theory and Practice. , 23-89 (1998).</li><li>Iliff, J. 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=A+Paravascular+Pathway+Facilitates+CSF+Flow+Through+the+Brain+Parenchyma+and+the+Clearance+of+Interstitial+Solutes,+Including+Amyloid+beta.">A Paravascular Pathway Facilitates CSF Flow Through the Brain Parenchyma and the Clearance of Interstitial Solutes, Including Amyloid beta.</a> Science Translational Medicine. 4 (147), (2012).</li><li>Michalke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese+speciation+using+capillary+electrophoresis-ICP-mass+spectrometry.">Manganese speciation using capillary electrophoresis-ICP-mass spectrometry.</a> Journal of Chromatography A. 1050 (1), 69-76 (2004).</li><li>Kuhn, R., Hofstetter-Kuhn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Capillary electrophoresis: Principles and practice. , (1993).</li><li>Michalke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoretic+methods+for+a+clear+identification+of+selenoamino+acids+in+complex+matrices+such+as+human+milk.">Capillary electrophoretic methods for a clear identification of selenoamino acids in complex matrices such as human milk.</a> Journal of Chromatography A. 716 (1-2), 323-329 (1995).</li><li>Yang, W. 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=Regulation+of+ferroptotic+cancer+cell+death+by+GPX4.">Regulation of ferroptotic cancer cell death by GPX4.</a> Cell. 156 (1-2), 317-331 (2014).</li><li>Shimada, K., Hayano, M., Pagano, N. C., Stockwell, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Line+Selectivity+Improves+the+Predictive+Power+of+Pharmacogenomic+Analyses+and+Helps+Identify+NADPH+as+Biomarker+for+Ferroptosis+Sensitivity.">Cell-Line Selectivity Improves the Predictive Power of Pharmacogenomic Analyses and Helps Identify NADPH as Biomarker for Ferroptosis Sensitivity.</a> Cell Chemical Biology. 23 (2), 225-235 (2016).</li></ol>

The authors have nothing to disclose.

Since iron plays a prominent role in OS progression, thus facilitating mitochondrial dysfunction or FTP, a versatile CE-ICP-MS based quantitative method for simultaneous Fe(II)/Fe(III) speciation is presented in this article and its application is exemplarily demonstrated in cell lysates. The method provided short analysis time and the figures of merit (LOQ, precision, recovery) are suitable for samples being relevant for iron redox speciation specifically in neurodegenerative and cancer research. Compared to previous methods based on LC, this CE-based method is practically independent of column batches and previously observed reproducibility problems after LC-column change. Capillary preparation before each run is &#60;4 minutes and analysis time per sample with moderate salinity up to 3 min. Apart from molecule charge and size, migration time in CZE depends on conductivity at the sample plug, which causes migration time variation or shifts when samples themselves influence conductivity considerably. Such shifts in migration time are well known in capillary electrophoresis. This is a CZE-immanent problem, known from literature21,22. Standards and SH-SY5Y cell lysates had moderate and homogenous conductivity. Consequently, migration times showed only little changes with good precision. For samples with high conductivity, however, prolonged migration times may be observed up to 5 min. Therefore, standard additions are recommended for clear species identification.
A critical issue in iron redox speciation is species stability (i.e., maintenance of Fe(II)/(III) equilibria) during sample preparation8,13. Inappropriate pH or chelating chemicals as well as inappropriate storage conditions such as oxygen (air) in contact with sample or a break in deep-frozen storage can easily change the Fe(II)/(III) balance. Therefore, for preparation of SH-SY5Y cell lysates, a lysis buffer was chosen without any chelating chemicals, physiological pH, but inert gas overlay during sample preparation, in sample containers and immediate deep freezing was applied for these samples.
In the literature, one can find semi-quantitative approaches to monitor Fe(II). For improved understanding of iron&#180;s role in oxidative stress, several research groups developed Fe(II)-specific probes to semi-quantitatively monitor and visualize aberrant elevation of ferrous iron in vitro. However, important to note, such probes do not consider Fe(III) and do not quantify but report just &#8220;more&#8221; or &#8220;less&#8221; Fe(II)). To date, only a few biomarkers are available to determine OS and FPT, being due to the lack of reliable methods to simultaneously quantify the Fe(II)/Fe(III) redox species23,24. Having this in mind, the presented method - facilitating fast quantification of both, Fe(III) and Fe(II) in one run - may become a promising tool to deepen the insight into iron-dependent molecular processes.

Today, it is most evident that iron-mediated oxidative stress (OS) plays a crucial role in multiple disorders specifically in neurodegenerative brain disorders, like Alzheimer&#8217;s and Parkinson&#8217;s disease as well as in cancer1,2,3,4. OS is closely related to the state and balance of the redox-couple Fe(II)/Fe(III). While Fe(III) is redox-inactive, Fe(II) potently generates reactive oxygen species (ROS) by catalyzing H2O2 decomposition followed from hydroxyl radical production and membrane lipid peroxidation5,6. On a molecular level, Fe(II)-generated ROS and peroxidized phospholipids are a strong attack to the integrity of proteins, lipids and DNA7,8. Such detrimental cellular dysfunction was demonstrated to induce mitochondrial dysfunction with decreased ATP-content9 and can even trigger a programmed necrotic cell death, known as ferroptosis (FPT)10,11. Therefore, quantitative Fe(II)/(III) redox speciation is of eminent importance in a broad spectrum of redox-related disorders.
Chemical speciation is a well-established tool for the study of trace elements biological role and metabolism in general7,8 as well as in neurodegenerative conditions12,13,14,15,16,17. Methods for Fe-redox speciation found in literature are typically based on liquid chromatography (LC) separation. Some of the literature use inductively coupled plasma mass spectrometry (ICP-MS) as an element selective detector. However, in routine LC work, excessive purge times were needed between runs. Even more problematic, batch-to-batch variation of LC columns forced re-optimization of the elution conditions after each column change. These problems are hampering high-throughput. Additional time is required to gain acceptable reliability and thoroughly evaluate the method again.
To circumvent these drawbacks, a method is presented here for Fe(II)/Fe(III) redox speciation based on capillary electrophoresis inductively coupled plasma mass spectrometry (CE-ICP-MS). CE offers various advantages compared to LC18. Capillaries have no stationary phase and thus depend (nearly) not on batch identity. When aged or blocked, they are replaced quickly, showing usually unchanged performance. The purge and cleaning steps between samples are effective and short, and the analysis time per sample is short, too.
The presented method is reliable with good figures of merit. As a proof-of-principle, the method is applied to human dopaminergic neuroblastoma (SH-SY5Y) cell lysate, a sample type important in neurodegeneration as well as cancer research19.

<table><tbody><tr><td>CE capillary</td><td>CS-Chromatographie Service, Langerwehe, Germany</td><td>105180-25</td><td></td></tr><tr><td>CE system</td><td>PrinCe technolgies</td><td>0005.263</td><td>model PrinCe 760</td></tr><tr><td>Conical Superclear Tubes 15 ml</td><td>Analytics-shop.com by Altmann Analytik</td><td>PEN0777704</td><td></td></tr><tr><td>Conical Superclear Tubes 50 ml</td><td>Analytics-shop.com by Altmann Analytik</td><td>PEN0777694</td><td></td></tr><tr><td>FeCl&lt;sub&gt;2&lt;/sub&gt; * 4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>103861</td><td></td></tr><tr><td>FeCl&lt;sub&gt;3&lt;/sub&gt;</td><td>Merck</td><td>803945</td><td></td></tr><tr><td>Fluidflex Silikon HG-Schlauch</td><td>ProLiquid</td><td>4001106HG</td><td></td></tr><tr><td>Fused silica capillary OD 360 &amp;micro;m, ID 50 &amp;micro;m</td><td>Chromatographie Service GmbH</td><td>105180-25</td><td></td></tr><tr><td>hydrochloric acid, 1 M</td><td>Merck</td><td>1101652500</td><td>corrosive</td></tr><tr><td>ICP-MS</td><td>Perkin Elmer</td><td>N814003</td><td></td></tr><tr><td>Luer, 3-way female</td><td>BioRad</td><td>7318229</td><td></td></tr><tr><td>Luer, cone male</td><td>neoLab Migge</td><td>2-1895</td><td></td></tr><tr><td>Luer, male</td><td>neoLab Migge</td><td>2-1880</td><td></td></tr><tr><td>Peakfit peak evaluation software</td><td>Systat</td><td>PeakFit 4.12</td><td></td></tr><tr><td>Pt-wire</td><td>Carl Roth</td><td>0737.1</td><td></td></tr><tr><td>PVC tube</td><td>ProLiquid</td><td>6000002</td><td></td></tr><tr><td>RIPA buffer</td><td>Abcam</td><td>ab156034</td><td></td></tr><tr><td>Tetramethylammoniumhydroxide, 25 %</td><td>Merck</td><td>814748</td><td>corrosive</td></tr><tr><td>TYGON-tube R-3607</td><td>ProLiquid</td><td>3700203A</td><td></td></tr></tbody></table>

setup of capillary electrophoresis-inductively coupled plasma mass spectrometry (ce-icp-ms) for quantification of iron redox species (fe(ii), fe(iii))

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative conditions. Excessive iron results in free redox-active Fe(II) and can cause devastating effects within the cell like oxidative stress (OS) and death by lipid peroxidation known as ferroptosis (FPT). Therefore, quantitative measurements of ferrous (Fe(II)) and ferric (Fe(III)) iron rather than total Fe-determination is the key for closer insight into these detrimental processes. Since Fe(II)/(III) determinations can be hampered by fast redox-state shifts and low concentrations in relevant samples, like cerebrospinal fluid (CSF), methods should be available that analyze quickly and provide low limits of quantification (LOQ). Capillary electrophoresis (CE) offers the advantage of fast Fe(II)/Fe(III) separation and works without a stationary phase, which could interfere with the redox balance or cause analyte sticking. CE combined with inductively coupled plasma mass spectrometry (ICP-MS) as a detector offers further improvement of detection sensitivity and selectivity. The presented method uses 20 mM HCl as a background electrolyte and a voltage of +25 kV. Peak shapes and concentration detection limits are improved by conductivity-pH-stacking. For reduction of 56[ArO]+, ICP-MS was operated in the dynamic reaction cell (DRC) mode with NH3 as a reaction gas. The method achieves a limit of detection (LOD) of 3 &#181;g/L. Due to stacking, higher injection volumes were possible without hampering separation but improving LOD. Calibrations related to peak area were linear up to 150 &#181;g/L. Measurement precision was 2.2% (Fe(III)) to 3.5% (Fe(II)). Migration time precision was &#60;3% for both species, determined in 1:2 diluted lysates of human neuroblastoma (SH-SY5Y) cells. Recovery experiments with standard addition revealed accuracy of 97% Fe(III) and 105 % Fe(II). In real-life bio-samples like CSF, migration time can vary according to varying conductivity (i.e., salinity). Thus, peak identification is confirmed by standard addition.

Measurements of standards and calibration 
Migration times were elucidated by single standard injections: the Fe(III) standard was monitored at 118 s of migration time and the Fe(II) standard at 136 s of migration time. Limits of detection were calculated using 3&#963; criterion referring to baseline noise and a standard concentration of 50 &#181;g/L. LOD(Fe(II) was 3.1 &#181;g/L and LOD(Fe(III) was 3.2 &#181;g/L. Peak area based calibration for both iron species was linear from the LOD to 150 &#181;g/L. While linearity of Fe(III) was proven also for higher concentration, the slope of calibration curve for Fe(II) decreased. An upper concentration limit of 150 &#181;g/L was considered to be sufficient since bio-samples relevant for Fe(II)/(III) determination typically have lower Fe concentration. In case of higher concentration, samples may be diluted accordingly. Peak-height calibration was checked up to 600 &#181;g/L and showed linearity over the whole tested range. This is shown in Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61055/61055fig2v2.jpg" /> 
Figure 2: Calibration curves (peak height) of Fe(III) and Fe(II).&#160;Peak height-related calibrations of both Fe redox species are linear with a slope of ca. 161 *X <a href="https://www.jove.com/files/ftp_upload/61055/61055fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Analysis of SH-SY5Y cell lysate 
The analysis of the SH-SY5Y cell lysate showed a slightly slower migration for iron redox species due to the somewhat higher conductivity. Fe(III) was monitored at 124 s of migration time, Fe(II) at 158 s of migration time. Migration time precision in SH-SY5Y cell lysate was 2% for Fe(III) and 3% for Fe(II). Quantitative Fe(II) and Fe(III) measurements using this method revealed Fe(III) concentration of 330 &#181;g/L and Fe(II) concentration 84 &#181;g/L, both resulting in a Fe(II)/Fe(III) ratio of 0.25. The respective 56Fe-selective electropherogram is demonstrated in Figure 3.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61055/61055fig3v2.jpg" /> 
Figure 3: 56Fe-specific electropherogram of SH-SY5Y cell lysate.&#160;Fe(III) is monitored at 123 s reaching 58025 cps peak height, being clearly separated from Fe(II) at 158 s, reaching 22800 cps <a href="https://www.jove.com/files/ftp_upload/61055/61055fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry CE-ICP-MS for Quantification of Iron Redox Species FeII, FeIII at JoVE.com

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry CE-ICP-MS for Quantification of Iron Redox Species FeII, FeIII

This iron redox speciation method is based on capillary electrophoresis-inductively coupled plasma mass spectrometry with sample stacking combined with short analysis in one run. The method quickly analyzes and provides low limits of quantification for iron redox species across a diverse range of tissues and biofluid samples.

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS) for Quantification of Iron Redox Species (Fe(II), Fe(III))

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative ...

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Chemistry

8.0K Views.  Helmholz Zentrum München-German Research Center for Environmental Health. Overmatig ijzer resulteert in gratis redox-actieve ijzer twee-plus die verwoestende effecten in de cel kan veroorzaken, zoals oxidatieve stress en celsterfgevallen door lipideperoxidatie bekend als ferroptose. Kwantitatieve metingen van ijzer en ijzer zijn de sleutel tot een beter inzicht in deze schadelijke processen. Deze CE-ICP-Ms-techniek biedt zowel:snelle analyse als lage kwantificeringslimieten, die beide nodig zijn voor het kwantificeren van ijzer-redoxsoorten.Begin met het sel...

Video: Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry CE-ICP-MS for Quantification of Iron Redox Species FeII, FeIII

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Biochemistry

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance (EPR) or ultraviolet-visible and near-infrared (UV-Vis-NIR) spectroscopies, we obtain useful information such as electron affinity, ionization potential, band-gap energies, the type of charge carriers, and degradation information that can be used to synthesize stable organic electronic devices. In this study, we present electrochemical and spectroelectrochemical methods to analyze the processes occurring in active layers of an organic device as well as the generated charge carriers.

In this article, we describe electrochemical, electron paramagnetic resonance, and ultraviolet-visible and near-infrared spectroelectrochemical methods to analyze organic compounds for application in organic electronics.

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence of at least one energetic pyrophosphate moiety on the myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, and cellular energy charge. Owing to the absence of a chromophore in these metabolites, a very high charge density, and low abundance, their analysis requires radioactive tracer, and thus it is convoluted and expensive. Here, the study presents a detailed protocol to perform absolute and high throughput quantitation of inositol pyrophosphates from mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables the sensitive profiling of all biologically relevant PP-InsPs species in mammalian cells, enabling baseline separation of regioisomers. Absolute cellular concentrations of PP-InsPs, including minor isomers, and monitoring of their temporal changes in HCT116 cells under several experimental conditions are presented.

A procedure for capillary electrophoresis electrospray ionization mass spectrometry for the absolute quantitation of inositol pyrophosphates from mammalian cell extracts is described.

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Bioengineering

Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the toxicokinetics of these compounds have both historical and modern-day relevance. Zebrafish have become an attractive model organism in elucidating pharmaco- and toxicokinetics in environmental exposure and clinical translation studies. Although zebrafish studies have the benefit of being higher-throughput than rodent models, there are several significant constraints to the model.
One such limitation is inherent in the waterborne dosing regimen. Water concentrations from these studies cannot be extrapolated to provide reliable internal dosages. Direct measurements of the metal-based compounds allow for a better correlation with compound-related molecular and biological responses. To overcome this limitation for metals and metal-based compounds, a technique was developed to digest zebrafish larval tissue after exposure and quantify metal concentrations within tissue samples by inductively coupled plasma mass spectrometry (ICPMS).
ICPMS methods were used to determine the metal concentrations of platinum (Pt) from cisplatin and ruthenium (Ru) from several novel Ru-based chemotherapeutics in zebrafish tissue. Additionally, this protocol distinguished concentrations of Pt that were sequestered in the chorion of the larval compared with the zebrafish tissue. These results indicate that this method can be applied to quantitate the metal dose present in larval tissues. Further, this method may be adjusted to identify specific metals or metal-based compounds in a broad range of exposure and dosing studies.

The increased rate of pharmaco- and toxicokinetic analyses of metals and metal-based compounds in zebrafish can be advantageous for environmental and clinical translation studies. The limitation of unknown waterborne exposure uptake was overcome by conducting trace metal analysis on digested zebrafish tissue using inductively coupled plasma mass spectrometry.

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.
The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.
A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 &#181;M. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...

Biology

Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The use of experimental animal models of iron overload or deficiency has been instrumental to advance knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis. The measurement of total iron levels in animal tissues is commonly performed with atomic absorption spectroscopy or with a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. For many years, the colorimetric assay has been used for the measurement of the non-heme iron content in a wide range of animal tissues. Unlike atomic absorption spectroscopy, it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells. Moreover, it does not require sophisticated analytical skills or highly expensive equipment, and can thus be easily implemented in most laboratories. Finally, the colorimetric assay can be either cuvette-based or adapted to a microplate format, allowing higher sample throughput. The present work provides a well-established protocol that is suited for the detection of alterations in tissue iron levels in a variety of experimental animal models of iron overload or iron deficiency.

Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories.

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The ...

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS) for Quantification of Iron Redox Species (Fe(II), Fe(III))

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