The research reported here was supported in part by the National Cancer Institute of the National Institutes of Health under award no. R33 CA217702 and the Initiative for Maximizing Student Development (IMSD) program. The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

Human serum specimens were obtained from consenting volunteers under IRB approval and according to the principles expressed in the Declaration of Helsinki. Specimens were coded and de-identified before transfer to the analytical laboratory.
1. Sample preparation
<ol>
	<li>HepG2 cell lysates
	<ol>
		<li>Seed about 10 x 106 HepG2 cells per flask in 16 T75 flasks with 14 mL of EMEM media supplemented with 10% fetal bovine serum (FBS) and grow cells for 2 days.</li>
		<li>Prepare RIPA buffer: in a 50 mL tube, add 1.5 mL of 5 M NaCl, 2.5 mL of 1 M Tris-HCl (pH = 7), 500 &#956;L of NP-40 reagent, then bring the final volume to 50 mL with DI water.</li>
		<li>Prepare lysis buffer: aliquot 20 mL of RIPA buffer into a 50 mL tube and add 200 &#956;L of a 100x protease inhibitor solution to inhibit protein and lipid degradation. Store at 4 &#176;C. 
		NOTE: Lysis buffer is compatible with TBARS reagents and does not interfere with absorbance at 532 nm. If planning to use a different lysis buffer or add additional ingredients to the lysis buffer, preliminary validation studies need to be done to verify that lysis buffer components are compatible with the TBARS assay.</li>
		<li>Remove media containing 10% FBS and wash cells 2x with 5 mL of cold, sterile 1x PBS.</li>
		<li>Add 1 mL of lysis buffer to the T75 flasks containing the cells and incubate them for 10 min at room temperature (RT) with constant swirling to ensure the buffer is well-distributed.</li>
		<li>Collect lysates into appropriately labeled 2 mL snap-cap polypropylene tubes and incubate on ice for 10 min.</li>
		<li>Spin the lysates at 5,000 x g for 10 min at RT to collect cell debris, and aspirate supernatants into a single 15 mL tube.</li>
		<li>Concentrate cell lysate supernatant four-fold using a Speed Vac at 50 &#176;C and 3 mbar and make aliquots of 94 &#956;L each into 2 mL snap-cap polypropylene tubes. Store samples at -80 &#176;C until they are used for in vitro oxidation and/or TBARS assay. 
		NOTE: To avoid concentrating the cell lysate&#160;supernatant, cells can also be detached using 3 mL of 1x trypsin, neutralized with 6 mL of media, and washed 2x with 5 mL of cold PBS. Cell pellets can then be reconstituted in 250 &#956;L of lysis buffer, and steps 1.1.6 and 1.1.7 can then be performed.</li>
		<li>Prepare a 35 mM CuCl2 stock solution in acetic acid (pH = 4).
		<ol>
			<li>Prepare acetic acid solution (pH = 4): Dilute 1 &#956;L of glacial acetic acid in 100 mL of DI water (pH should be approximately 4 but confirm this with a pH meter). Add more water or acetic acid to adjust the pH to 4.</li>
			<li>Weight out about 0.1936 g of copper II chloride and dissolve in 10 mL of the acetic acid solution (pH = 4) to make a 144 mM CuCl2 stock. Aliquot 490 &#956;L from this solution and add to 1,510 &#956;L of acetic acid (pH = 4) to make a 35 mM CuCl2 solution.</li>
		</ol>
		</li>
		<li>Aliquot 6 &#956;L from the 35 mM CuCl2 stock solution and add it to six samples containing 94 &#956;L of cell lysate to make a final CuCl2 concentration of about 2 mM. Add 6 &#956;L of an acetic acid solution (pH = 4) that does not have CuCl2 to six samples containing 94 &#956;L of cell lysates to use as controls. The final volume of cell lysate should be 100 &#956;L, which is what will be used for the TBARS assay. 
		NOTE: Making the 35 mM CuCl2 stock solution in acetic acid (pH = 4) is necessary to prevent precipitation of copper hydroxide.</li>
		<li>Incubate samples in an oven at 37 &#176;C for 24 h and perform a TBARS assay on each sample containing a final volume of 100 &#956;L.</li>
		<li>Repeat steps 1.1.9 and 1.1.11 2x on separate days to check the reproducibility of the TBARS assay for HepG2 cell lysates.</li>
	</ol>
	</li>
	<li>Low density lipoproteins 
	NOTE: Typically, pre-purified low density lipoprotein (LDL) contains some amount of EDTA. LDL samples used here contain 0.01% EDTA. EDTA can inhibit the in vitro Cu(II)-mediated oxidation of LDL. Hence, it may be necessary to remove EDTA from LDL samples prior to experiments or analysis. Steps 1.2.1&#8211;1.2.5 describe this process. 
	CAUTION: Sodium hydroxide is corrosive and causes irritation in skin and eyes. Use proper personal protective equipment.
	<ol>
		<li>Aliquot 24 &#956;L from a 5.51 mg/mL LDL stock (protein concentration determined by modified Lowry method using BSA as a standard) into appropriately labeled 1 mL snap-cap polypropylene tubes. Make as many aliquots as needed and store at 4 &#176;C until use in oxidation and/or TBARS assay.</li>
		<li>Prepare a 10 mM HEPES buffer in 0.15 M NaCl adjusted to pH = 7 with NaOH beads: dissolve 4.39 g of NaCl in 0.49 L of water, then add 1.19 g of HEPES. Dissolve well with a stir bar. Add sodium hydroxide beads until pH is 7. Dilute to 0.5 L with water. Store buffer at 4 &#176;C and use within 3 months.</li>
		<li>Add 476 &#956;L of the 10 mM HEPES buffer in 0.15 M NaCl (pH = 7) to the aliquoted LDL samples to bring final volume to 500 &#956;L. Add diluted LDL sample to a 0.5 mL centrifugal spin filter device with a 100K molecular weight cutoff.</li>
		<li>Spin samples at 14,000 x g for 10 min at RT, leaving a final retentate volume of about 30 &#956;L. Reconstitute samples in 480 &#956;L of the 10 mM HEPES buffer in 0.15 M NaCl (pH = 7) and spin again at 14,000 x g for 10 min at RT. Perform this step 2x for a total of four spin-throughs.</li>
		<li>Place filter device upside down into a new 2 mL snap-cap polypropylene tube, and centrifuge at 1000 x g for 2 min to collect LDL sample (final volume = about 30 &#956;L).</li>
		<li>Aliquot sample into appropriately labeled 1 mL tube and add 20 &#956;L of water to each sample to achieve a final volume of 50 &#956;L.</li>
		<li>Preparation of 200 &#956;M CuCl2 stock solution in acetic acid (pH = 4)
		<ol>
			<li>Prepare acetic acid solution (pH = 4): see step 1.1.9.1.</li>
			<li>Prepare a 144 mM CuCl2 stock solution (see step 1.1.9.2), then aliquot 5.5 &#956;L from the 144 mM CuCl2 stock and dissolve in a final volume of 4 mL of acetic acid (pH = 4) to make the 200 &#956;M solution.</li>
		</ol>
		</li>
		<li>Aliquot 2.7 &#956;L from the 200 &#956;M CuCl2 stock solution and add it to six samples containing 50 &#956;L of LDL to achieve a final CuCl2 concentration of ~10 &#956;M. Add 2.7 &#956;L from an acetic acid solution (pH = 4) that does not contain CuCl2 to six samples containing 50 &#956;L of LDL to be used for the controls.</li>
		<li>Incubate LDL samples for 2 h in an oven at 37 &#176;C. After 2 h, bring the final volume to 100 &#956;L for each sample with 10 mM HEPES buffer in 0.15 M NaCl (pH = 7). Immediately perform a TBARS assay.</li>
		<li>Repeat steps 1.2.3&#8211;1.2.9 2x on two different days to test reproductivity of the TBARS assay.</li>
	</ol>
	</li>
	<li>Human serum
	<ol>
		<li>From a human serum sample, make aliquots of 94 &#956;L each into 2 mL snap-cap polypropylene tubes and store samples at -80 &#176;C.</li>
		<li>Prepare a 35 mM CuCl2 stock solution in acetic acid (pH = 4): see step 1.1.9.</li>
		<li>Aliquot 6 &#956;L from the CuCl2 stock solution and add it to six samples containing 94 &#956;L of human serum to make a final CuCl2 concentration of about 2 mM. Add 6 &#956;L of an acetic acid solution (pH = 4) that does not have CuCl2 to six samples containing 94 &#956;L of human serum to use as controls.</li>
		<li>Incubate human serum samples for 24 h in an oven at 37 &#176;C and determine levels of oxidation with TBARS assay (section 4).</li>
		<li>Repeat steps 1.3.2&#8211;1.3.4 2x on two separate days to determine reproducibility of the TBARS assay.</li>
	</ol>
	</li>
</ol>
2. Reagent preparation
CAUTION: Thiobarbituric acid causes skin and eye irritation and maybe harmful by inhalation or skin absorption. Acetic acid can damage internal organs if inhaled. Prepare all acid solutions in a fume hood.
<ol>
	<li>Preparation of 8.1% (w/v) sodium dodecylsulfate (SDS) solution
	<ol>
		<li>Weight out 32.4 g of SDS and dissolve in 350 mL of DI water in a beaker. Use a magnetic stir bar to gently dissolve SDS and avoid making bubbles. Bring final volume to 400 mL with DI water and store SDS solution at RT. 
		NOTE: Here, excess 8.1% SDS solution is prepared; however, for 96 samples, only about 20 mL of the 8.1% SDS solution are needed. Prepare this solution according to the number of samples being analyzed.</li>
	</ol>
	</li>
	<li>Preparation of 3.5 M sodium acetate buffer (pH = 4)
	<ol>
		<li>Dilute 100 mL of glacial acetic acid in 350 mL of DI water in a beaker. Use a magnetic stir bar to gently dissolve it.</li>
		<li>Prepare a 6.5 M NaOH solution using sodium hydroxide beads in water. Dissolve 13 g of NaOH beads in 40 mL of DI water and bring to a final volume of 50 mL with DI water.</li>
		<li>Slowly add about 46 mL of the 6.5 M NaOH solution to the acetic acid solution while mixing with the stir bar (this should raise the pH to 4, but confirm by slowly adding the NaOH solution while measuring using a pH meter).</li>
		<li>Bring final volume to 500 mL with DI water and store sodium acetate buffer at RT.</li>
	</ol>
	</li>
	<li>Preparation of 0.8% aqueous solution of thiobarbituric acid (adjusted to pH = 4) 
	NOTE: In this step, preparation of thiobarbituric acid is optimized for large volumes, since a large number of samples is going to be analyzed (108 samples, not including the standards). Prepare this solution depending on the number of samples planned for analysis.
	<ol>
		<li>Prepare a 5 M sodium hydroxide solution using sodium hydroxide beads and water: dissolve 4 g of sodium hydroxide beads in a final volume of 20 mL of water. Store in a plastic container. This solution should be freshly prepared for each batch.</li>
		<li>Weight 4 g of thiobarbituric acid and add 450 mL of DI water. Use a magnetic stir bar to gently dissolve it. 
		NOTE: This solution will eventually be brought to a 500 mL total volume.</li>
		<li>While dissolving thiobarbituric acid with a stir bar, add (slowly and in a dropwise manner) about 3 mL of the 5 M NaOH solution in 100 &#956;L increments. After adding the NaOH solution, the thiobarbituric acid particles will start to dissolve.</li>
		<li>If the thiobarbituric acid particles still have not fully dissolved, add more of the 5 M NaOH solution in 100 &#956;L increments until all thiobarbituric acid particles are fully dissolved. For this particular volume of solution, a total of 4 mL of the 5 M NaOH solution is added to fully dissolve the thiobarbituric acid particles. 
		NOTE: At this concentration, thiobarbituric acid will not fully dissolve unless the pH is nearly 4.</li>
		<li>Stop adding NaOH after all the thiobarbituric&#160;acid has fully dissolved. Avoid exceeding a pH of 4. The final pH can be verified by taking 1 &#956;L from the mixing thiobarbituric&#160;acid solution and placing it onto pH paper.</li>
		<li>Bring final volume to 500 mL with DI water and store aqueous 0.8% thiobarbituric acid solution at RT.</li>
	</ol>
	</li>
</ol>
3. Malondialdehyde bis(dimethyl acetal) standard sample preparation
NOTE: Malondialdehyde (MDA) is unstable and not commercially available. However, there are different chemical forms of MDA that are commercially available, such as MDA tetrabutylammonium salt, MDA bis(dimethyl acetal), and MDA bis(diethyl acetal). Of these three chemical forms, MDA bis(dimethyl acetal) is used here, because a majority of studies use this same standard21,22. If choosing to use the other two chemical forms of MDA, prior validation of their suitability should be carried out.
<ol>
	<li>Prepare a 550 &#956;M MDA bis(dimethyl acetal) stock solution by diluting 92 &#956;L of pure MDA bis(dimethyl acetal) in 1 L of DI water. Use a magnetic stir bar to mix the solution thoroughly for 10 min. Store solution at 4 &#176;C and use within 1 month.</li>
	<li>Prepare a 200 &#956;M MDA bis(dimethyl acetal) by diluting 726 &#956;L from the 550 &#956;M MDA bis(dimethyl acetal) stock in 1274 &#956;L of DI water. This 200 &#956;M MDA bis(dimethyl acetal) solution should be prepared fresh every time a TBARS assay is performed.</li>
	<li>Standard curve preparation: take eight 2 mL snap-cap polypropylene tubes and label them with letters A through H. Add MDA bis(dimethyl acetal) from the 200 &#956;M stock and dilute in water as described in Table 1.</li>
	<li>Take eight glass tubes (13 mm x 100 mm) and label them A&#8211;H, then add 100 &#956;L of standard to the corresponding tubes. Perform six replicates for the blank standard (sample A) to calculate the limits of detection of the TBARS assay. 
	NOTE: The protocol can be paused here for no more than 1 h.</li>
</ol>
4. TBARS assay
NOTE: Once the TBARS assay is started, it should be finished without stopping.
<ol>
	<li>Take as many glass tubes as needed for the number of samples to be analyzed and label them with the names of the samples. Then, add 100 &#956;L of each prepared sample (as described above) to each glass tube.</li>
	<li>Add 200 &#956;L of 8.1% SDS to each sample and standard and gently swirl the glass tube in a circular motion to mix the sample.</li>
	<li>Add 1.5 mL of the 3.5 M sodium acetate buffer (pH = 4) to each sample and standard.</li>
	<li>Add 1.5 mL of the aqueous 0.8% thiobarbituric acid solution (pH = 4) to each sample and standard.</li>
	<li>Bring the final volume to 4 mL for each sample and standard by adding 700 &#956;L of DI water.</li>
	<li>Tightly cap each glass tube and incubate in a heating block set to 95 &#176;C for 1 h. Cover the glass tubes with aluminum foil to prevent condensation at the tops of the tubes.</li>
	<li>Remove the glass tubes from the heating block and incubate on ice for 30 min.</li>
	<li>Centrifuge samples and standards at 1500 x g for 10 min at 4 &#176;C. After centrifugation, keep the glass tubes containing the samples and standards at RT. 
	NOTE: Keeping the samples on ice or at 4 &#176;C will cause the entire sample or standard to precipitate.</li>
	<li>Immediately after centrifugation, aliquot 150 &#956;L of supernatant from each tube and place into a separate well of a 96 well plate.</li>
	<li>Remove any bubbles from each well using a pipette tip. 
	NOTE: The presence of bubbles will yield inconsistent absorbance readings, leading to high assay imprecision.</li>
	<li>Read absorbances at 532 nm. Subtract the average absorbance reading of the blank samples from all other absorbance readings.</li>
	<li>Create a standard curve by plotting the blank-subtracted absorbance readings at 532 nm vs. the known concentration of each standard. Fit the data points using linear regression. Calculate unknown sample concentrations by using the equation of the linear regression line obtained from the standard curve.</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling,+standardization,+and+calibration.">Sampling, standardization, and calibration.</a> Fundamentals of Analytical Chemistry. 9th ed. , 153-196 (2014).</li><li>Skoog, D. A., Holler, F. J., Crouch, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction.">Introduction.</a> Principles of Instrumental Analysis. 6th ed. , 1-24 (2007).</li><li>Seibig, S., Van Eldik, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+[FeII(edta)]+Oxidation+by+Molecular+Oxygen+Revisited.+New+Evidence+for+a+Multistep+Mechanism.">Kinetics of [FeII(edta)] Oxidation by Molecular Oxygen Revisited. New Evidence for a Multistep Mechanism.</a> Inorganic Chemistry. 36 (18), 4115-4120 (1997).</li><li>Jeffs, J. W., 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=Delta-S-Cys-Albumin:+A+Lab+Test+that+Quantifies+Cumulative+Exposure+of+Archived+Human+Blood+Plasma+and+Serum+Samples+to+Thawed+Conditions.">Delta-S-Cys-Albumin: A Lab Test that Quantifies Cumulative Exposure of Archived Human Blood Plasma and Serum Samples to Thawed Conditions.</a> Molecular &amp; Cellular Proteomics. 18 (10), 2121-2137 (2019).</li><li>Yagi, K., Armstrong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Assay+for+the+Level+of+Total+Lipid+Peroxides+in+Serum+or+Plasma.">Simple Assay for the Level of Total Lipid Peroxides in Serum or Plasma.</a> Free Radical and Antioxidant Protocols. Methods in Molecular Biology. , 101-106 (1998).</li><li>Bernheim, F., Bernheim, M. L. C., Wilbur, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reaction+between+thiobarbituric+acid+and+the+oxidation+products+of+certain+lipides.">The reaction between thiobarbituric acid and the oxidation products of certain lipides.</a> Journal of Biological Chemistry. 174 (1), 257-264 (1948).</li><li>Wilbur, K. M., Bernheim, F., Shapiro, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thiobarbituric+acid+reagent+as+a+test+for+the+oxidation+of+unsaturated+fatty+acids+by+various+agents.">The thiobarbituric acid reagent as a test for the oxidation of unsaturated fatty acids by various agents.</a> Archives of Biochemistry. 24 (2), 305-313 (1949).</li><li>Kwon, T. W., Watts, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+malonaldehyde+by+ultraviolet+spectrophotometry.">Determination of malonaldehyde by ultraviolet spectrophotometry.</a> Journal of Food Science. 28 (6), 627-630 (1963).</li><li>Esterbauer, H., Schaur, F. J., Zollner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+and+biochemistry+of+4-hydroxynonenal,+malonaldehyde+and+related+aldehydes.">Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.</a> Free Radical Biology &amp; Medicine. 11 (1), 81-128 (1991).</li><li>Dalle-Donne, I., Rossi, R., Colombo, R., Giustarini, D., Milzani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+oxidative+damage+in+human+disease.">Biomarkers of oxidative damage in human disease.</a> Clinical Chemistry. 52 (4), 601-623 (2006).</li><li>Jentzsch, A. M., Bachmann, H., F&#252;rst, P., Biesalski, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+analysis+of+malondialdehyde+in+human+body+fluids.">Improved analysis of malondialdehyde in human body fluids.</a> Free Radical Biology and Medicine. 20 (2), 251-256 (1996).</li><li>Jo, C., Ahn, D. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorometric+Analysis+of+2-Thiobarbituric+Acid+Reactive+Substances+in+Turkey.">Fluorometric Analysis of 2-Thiobarbituric Acid Reactive Substances in Turkey.</a> Poultry Science. 77 (3), 475-480 (1998).</li><li>Tsikas, D., 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=Development,+validation+and+biomedical+applications+of+stable-isotope+dilution+GC-MS+and+GC-MS/MS+techniques+for+circulating+malondialdehyde+(MDA)+after+pentafluorobenzyl+bromide+derivatization:+MDA+as+a+biomarker+of+oxidative+stress+and+its+relation+to+15(S)-8-iso-prostaglandin+F2&#945;+and+nitric+oxide+(NO).">Development, validation and biomedical applications of stable-isotope dilution GC-MS and GC-MS/MS techniques for circulating malondialdehyde (MDA) after pentafluorobenzyl bromide derivatization: MDA as a biomarker of oxidative stress and its relation to 15(S)-8-iso-prostaglandin F2&#945; and nitric oxide (NO).</a> Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 1019, 95-111 (2016).</li><li>Barden, A. E., Mas, E., Croft, K. D., Phillips, M., Mori, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimizing+artifactual+elevation+of+lipid+peroxidation+products+(F+2-isoprostanes)+in+plasma+during+collection+and+storage.">Minimizing artifactual elevation of lipid peroxidation products (F 2-isoprostanes) in plasma during collection and storage.</a> Analytical Biochemistry. 449 (1), 129-131 (2014).</li><li>Jeffs, J. W., Ferdosi, S., Yassine, H. N., Borges, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+instability+of+glycated+albumin:+A+role+for+autoxidative+glycation.">Ex vivo instability of glycated albumin: A role for autoxidative glycation.</a> Archives of Biochemistry and Biophysics. 629, 36-42 (2017).</li><li>Lee, 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=Malondialdehyde+in+Stored+Plasma.">Malondialdehyde in Stored Plasma.</a> Biochemical and Biophysical Research Communications. 95 (4), 1663-1672 (1980).</li><li>Tsikas, D., 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=Simultaneous+GC-MS/MS+measurement+of+malondialdehyde+and+4-hydroxy-2-nonenal+in+human+plasma:+Effects+of+long-term+L-arginine+administration.">Simultaneous GC-MS/MS measurement of malondialdehyde and 4-hydroxy-2-nonenal in human plasma: Effects of long-term L-arginine administration.</a> Analytical Biochemistry. 524, 31-44 (2017).</li></ol>

The authors have no competing financial interests or other conflicts of interest to disclose.

Despite its limitations1,3,4,7,8,9,10,12,13,14,15,19 and a lack of suitability for comparison between laboratories, t...

Lipid peroxidation is a process in which free radicals, such as reactive oxygen species and reactive nitrogen species, attack carbon-carbon double bonds in lipids, a process that involves the abstraction of a hydrogen from a carbon and insertion of an oxygen molecule. This process leads to a mixture of complex products including, lipid peroxyl radicals, and hydroperoxides as the primary products, as well as malondialdehyde (MDA) and 4-hydroxynonenal as predominant secondary products1.
MDA has been widely used in biomedical research as a marker of lipid peroxidation due to its facile reaction with thiobarbituric acid (TBA). The reaction leads to the formation of MDA-TBA2, a conjugate that absorbs in the visible spectrum at 532 nm and produces a red-pink color2. Other molecules derived from lipid peroxidation besides MDA can also react with TBA and absorb light at 532 nm, contributing to the overall absorption signal that is measured. Similarly, MDA can react with most other major classes of biomolecules, potentially limiting its accessibility for reaction with TBA3,4. As such, this traditional assay is simply considered to measure &#8220;thiobarbituric acid reactive substances&#8221; or TBARS5.
When correctly applied and interpreted, the TBARS assay is generally considered a good indicator of the overall levels of oxidative stress in a biological sample6. Unfortunately, as documented by Khoubnasabjafari and others, the TBARS assay is often conducted and interpreted in ways that facilitate dubious conclusions3,4,7,8,9,10,11. The causes for this are rooted primarily in sample-related pre-analytical variables and a lack of assay ruggedness that prohibits seemingly minor variations in assay protocol without substantial changes in assay results1,7,12,13.
Preanalytical variables related to biospecimen handling and storage (e.g., blood plasma kept temporarily at -20 &#176;C)14,15 can have a major impact on TBARS assay results16,17; so much so, that TBARS assay results should not be compared across different laboratories unless warranted by explicit interlaboratory analytical validation data. This recommendation is akin to how western blots are commonly used and interpreted. Comparisons of band densities are valid for within-blot and perhaps within-laboratory studies, but comparing band densities between laboratories is generally considered an invalid practice.
Some researchers have suggested that MDA as measured by the TBARS assay simply does not meet the analytical or clinical criteria required of an acceptable biomarker3,9,10,18,19. Indeed, if the assay had not been developed over 50 years ago, it probably would not have gained the widespread use and tacit acceptability that it has today. Although there are other assays with greater analytical sensitivity, specificity, and ruggedness used for determining oxidative stress, TBARS assay based on absorbance at 532 nm remains by far one of the most commonly used assays for the determination of lipid peroxidation20, and thereby assessment of oxidative stress.
The TBARS assay can only be found as an expensive kit (over 400 U.S. dollars), in which the instructions do not provide detailed information on most concentrations of the reagents used. Additionally, the reagents provided can only be used for one experiment, because only one colorimetric standard curve can be made per kit. This can be problematic for researchers who intend to determine levels of oxidation within a few samples at different timepoints, because the same standard curve cannot be used at multiple times. Hence, multiple kits need to be purchased for multiple experiments. Currently, unless an expensive kit is purchased, there is not a detailed protocol available for how to perform a TBARS assay. Some researchers in the past have vaguely described how to perform a TBARS assay21,22, but neither a fully detailed protocol or comprehensive video on how to conduct the TBARS assay without an expensive kit is available in the literature.
Here we report a detailed, analytically validated for-purpose methodology on how to perform a TBARS assay in a simple, reproducible, and inexpensive way. Changes in the lipid peroxidation of human serum, HepG2 lysates, and low density lipoproteins upon treatment with Cu(II) ions are demonstrated as illustrative applications for the TBARS assay. Results demonstrate that this TBARS assay is consistent and reproducible on a day-to-day basis.

<table>
	<tbody>
		<tr>
			<td>1x Sterile PBS pH 7.4 1 L</td>
			<td>VWR, PA</td>
			<td>101642--262</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>50 mL self-standing centrifuge tube</td>
			<td>Corning, NY</td>
			<td>CLS430897</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>96 well plate, Non-Treated, clear, with lid, Non-sterile</td>
			<td>Thermo Fisher Scientific, MA</td>
			<td>280895</td>
			<td>To measure absorbance</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 100 kD centrifugal spin filter device</td>
			<td>Fisher Scientific, NH</td>
			<td>UFC510024</td>
			<td>LDL purification</td>
		</tr>
		<tr>
			<td>Caps for glass tubes</td>
			<td>Thermo Fisher Scientific, MA</td>
			<td>14-930-15D</td>
			<td>for TBARS assay</td>
		</tr>
		<tr>
			<td>Copper II Chloride</td>
			<td>SIGMA, MO</td>
			<td>222011-250G</td>
			<td>to induce oxidation</td>
		</tr>
		<tr>
			<td>Culture tubes, Disposable, with Screw-Cap Finish, Borosilicate Glass (13 x 100 mm)</td>
			<td>VWR, PA</td>
			<td>53283-800</td>
			<td>for TBARS assay</td>
		</tr>
		<tr>
			<td>Eagle&#39;s Minimum Essential Medium (EMEM)</td>
			<td>ATCC, VA</td>
			<td>HB-8065</td>
			<td>HepG2 cell media</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 1.5 mL</td>
			<td>eppendorf, NY</td>
			<td>22363204</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 2.0 mL</td>
			<td>Genesee Sceitific, CA</td>
			<td>22363352</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum US Source</td>
			<td>Omega Scientific, CA</td>
			<td>FB-11</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>SIGMA, MO</td>
			<td>27225-1L-R</td>
			<td>TBARS Reagent</td>
		</tr>
		<tr>
			<td>Halt Protease Inhibitor Cocktail (100x)</td>
			<td>Thermo Scientific, MA</td>
			<td>87786</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SIGMA, MO</td>
			<td>H3375-250G</td>
			<td>LDL solvent</td>
		</tr>
		<tr>
			<td>HepG2 Cells</td>
			<td>ATCC, VA</td>
			<td>HB-8065</td>
			<td>Biological matrix prototype</td>
		</tr>
		<tr>
			<td>Hydrocloric acid (HCl)</td>
			<td>Fisher Scientific, NH</td>
			<td>A144-212</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>Legend Micro 17 Centrifuge</td>
			<td>Thermo Scientific, MA</td>
			<td>75002431</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>Low Density Lipoprotein, Human Plasma</td>
			<td>Athens Research &#38; Technology, GA</td>
			<td>12-16-120412</td>
			<td>Biological matrix prototype</td>
		</tr>
		<tr>
			<td>Magnetic Stir Bars, Octagon 6-Assortment</td>
			<td>VWR, PA</td>
			<td>58948-025</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>Malondialdehyde bis (dimethyl acetal)</td>
			<td>SIGMA, MO</td>
			<td>8207560250</td>
			<td>TBARS Standard</td>
		</tr>
		<tr>
			<td>Multiskan Go Microplate Spectrophotometer</td>
			<td>Fisher Scientific, NH</td>
			<td>51119200</td>
			<td>To measure absorbance</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>EMD Millipore Corp, MA</td>
			<td>492016-100ML</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>SIGMA, MO</td>
			<td>S7653-1KG</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>SIGMA, MO</td>
			<td>436143-100G</td>
			<td>TBARS Reagent</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>SIGMA, MO</td>
			<td>367176-2.5KG</td>
			<td>TBARS Reagent</td>
		</tr>
		<tr>
			<td>SpeedVac Concentrator</td>
			<td>Thermo Scientific, MA</td>
			<td>SC250EXP</td>
			<td>For concentrating cell lysates</td>
		</tr>
		<tr>
			<td>T-75 Flask, Tissue Culture Treated, 250 mL, w/filter cap</td>
			<td>USA Scientific, FL</td>
			<td>658175</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Thiobarbituric Acid</td>
			<td>SIGMA, MO</td>
			<td>T5500-100G</td>
			<td>TBARS Reagent</td>
		</tr>
		<tr>
			<td>TRIS base</td>
			<td>Fluka, GA</td>
			<td>93362</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>Trypsin (1x)</td>
			<td>VWR, PA</td>
			<td>16777-166</td>
			<td>To detach HepG2 cells</td>
		</tr>
	</tbody>
</table>

evaluation of oxidative stress in biological samples using the thiobarbituric acid reactive substances assay

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 &#176;C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 &#956;M can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.

Under acidic conditions (pH = 4) and at 95 &#176;C, malondialdehyde (MDA) bis(dimethyl acetal) yields MDA23. MDA and closely related chemical congeners react with two molecules of thiobarbituric acid (TBA) to produce compounds called thiobarbituric acid reactive substances (TBARS), which give a red-pink color and have an absorbance &#955;max at 532 nm (Figure 1, Figure 2). Using MDA bis (dimethyl acetal) as the standard, standa...

Watch this Scientific Journal Video about Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay at JoVE.com

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

The goal of the thiobarbituric acid reactive substances assay is to assess oxidative stress in biological samples by measuring the production of lipid peroxidation products, primarily malondialdehyde, using visible wavelength spectrophotometry at 532 nm. The method described here can be applied to human serum, cell lysates, and low density lipoproteins.

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic ...

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27.2K Views.  Arizona State University. The goal of the thiobarbituric acid reactive substances assay is to assess oxidative stress in biological samples by measuring the production of lipid peroxidation products, primarily malondialdehyde, using visible wavelength spectrophotometry at 532 nm. The method described here can be applied to human serum, cell lysates, and low density lipoproteins.

Video: Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

Supercritical Nitrogen Processing for the Purification of Reactive Porous Materials

Supercritical fluid extraction and drying methods are well established in numerous applications for the synthesis and processing of porous materials. Herein, nitrogen is presented as a novel supercritical drying fluid for specialized applications such as in the processing of reactive porous materials, where carbon dioxide and other fluids are not appropriate due to their higher chemical reactivity. Nitrogen exhibits similar physical properties in the near-critical region of its phase diagram as compared to carbon dioxide: a widely tunable density up to ~1 g ml-1, modest critical pressure (3.4 MPa), and small molecular diameter of ~3.6 &#197;. The key to achieving a high solvation power of nitrogen is to apply a processing temperature in the range of 80-150 K, where the density of nitrogen is an order of magnitude higher than at similar pressures near ambient temperature. The detailed solvation properties of nitrogen, and especially its selectivity, across a wide range of common target species of extraction still require further investigation. Herein we describe a protocol for the supercritical nitrogen processing of porous magnesium borohydride.

Nitrogen is an effective supercritical fluid for extraction or drying processes due to its small molecular size, high density in the near-liquid supercritical regime, and chemical inertness. We present a supercritical nitrogen drying protocol for the purification treatment of reactive, porous materials.

Supercritical fluid extraction and drying methods are well established in numerous applications for the synthesis and processing of porous materials. ...

Using Polystyrene-block-poly(acrylic acid)-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are encapsulated in polystyrene-block-poly(acrylic acid) (PSPAA) shells and then used as monomers for their self-assembly. Spherical PSPAA micelles upon acid treatment are known to assemble into cylindrical micelles. Exploiting this tendency, the core-shell nanoparticles are induced to aggregate, coalesce, and then transform into long chains. When more than one type of nanoparticles are used, random and block &#8220;copolymers&#8221; of nanoparticles can be obtained. Detailed procedures are reported for the PSPAA encapsulation of nanoparticles, homo- and co-polymerization of the core-shell nanoparticles, separation and purification of the resulting nanoparticle chains. Transformations of single-line chains into double- and triple-line chains are also presented. The synergy between the polymer shell and the embedded nanoparticles leads to an unusual chain-growth polymerization mode, giving long nanoparticle chains that are distinct from the products of the traditional step-growth aggregation process.

Using Polystyrene-block-polyacrylic acid-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We report protocols for &#8220;polymerizing&#8221; various types of polymer-encapsulated metal nanoparticles into long chains of &#8220;homo-&#8220; and &#8220;co-polymers&#8221;.

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are ...

Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide (CdSe@CdS) nanorod photocatalysts. Seeded rods are grown using a colloidal hot-injection method and then moved to an aqueous medium by ligand exchange. CdSe@CdS nanorods, an iridium precursor and other salts are mixed and illuminated. The deposition process is initiated by absorption of photons by the semiconductor particle, which results with formation of charge carriers that are used to promote redox reactions. To insure photochemical oxidative growth we used an electron scavenger. The photogenerated holes oxidize the iridium precursor, apparently in a mediated oxidative pathway. This results in the growth of high quality crystalline iridium oxide particles, ranging from 0.5 nm to about 3 nm, along the surface of the rod. Iridium oxide grown on CdSe@CdS heterostructures was studied by a variety of characterization methods, in order to evaluate its characteristics and quality. We explored means for control over particle size, crystallinity, deposition location on the CdS rod, and composition. Illumination time and excitation wavelength were found to be key parameters for such control. The influence of different growth conditions and the characterization of these heterostructures are described alongside a detailed description of their synthesis. Of significance is the fact that the addition of iridium oxide afforded the rods astounding photochemical stability under prolonged illumination in pure water (alleviating the requirement for hole scavengers).

A protocol for the photochemical oxidative growth of small crystalline iridium oxide nanoparticles on the surface of CdSe@CdS seeded rod nanoparticles is presented.

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide ...

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.

A protocol is presented for simultaneously extracting metabolites, proteins, and lipids from a single soil sample, allowing reduced sample preparation times and enabling multi-omic mass spectrometry analyses of samples with limited quantities.

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and ...

Separation of Aldehydes and Reactive Ketones from Mixtures Using a Bisulfite Extraction Protocol

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer by generating a charged structure provides an opportunity to use extraction as a simple purification technique. By combining the use of a miscible organic solvent with saturated sodium bisulfite, aldehydes and reactive ketones can be successfully transformed into charged bisulfite adducts that can then be separated from other organic components of a mixture by the introduction of an immiscible organic layer. Here, we describe a simple protocol for the removal of aldehydes, including sterically-hindered neopentyl aldehydes and some ketones, from chemical mixtures. Ketones can be separated if they are sterically unhindered cyclic or methyl ketones. For aliphatic aldehydes and ketones, dimethylformamide is used as the miscible solvent to improve removal rates. The bisulfite addition reaction can be reversed by basification of the aqueous layer, allowing for the re-isolation of the reactive carbonyl component of a mixture.

Here, we present a protocol to remove aldehydes and reactive ketones from mixtures by a liquid-liquid extraction protocol directly with saturated sodium bisulfite in a miscible solvent. This combined protocol is rapid and facile to perform. The aldehyde or ketone can be re-isolated by the basification of the aqueous layer.

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer ...

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The resulting devices are an example of self-propelled objects capable of generating motion without external actuation and have potential applications in medicine and environmental sciences for a variety of purposes ranging from micro-stirring, targeted therapeutic delivery, to water remediation (e.g., cleaning oil spills). This method employs reactive inkjet printing to generate well-defined small-scale solid silk structures by converting water soluble regenerated silk fibroin (silk I) to insoluble silk fibroin (silk II). These structures are also selectively doped in specific regions with the enzyme catalase in order to produce motion via bubble generation and detachment. The number of layers printed determines the three-dimensional (3D) structure of the device, and so here the effect of this parameter on the propulsive trajectories is reported. The results demonstrate the ability to tune the motion by varying the dimensions of the printed structures.

This protocol demonstrates the ability to utilize reactive inkjet printing to print self-motile biocompatible and environmentally friendly micro-stirrers for use in biomedical and environmental applications.

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...