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

All procedures described in the protocol related to animal or human samples/tissues were approved by and followed the institutional guidelines of the human and animal research ethics committee.

1. Sample homogenization/lysis

1. Frozen tissue samples
   1. Mince frozen tissue (~30 mg) on a glass microscope slide on dry ice using a prechilled razor blade and forceps. Transfer the minced tissue to a prechilled 5 mL round-bottom polystyrene tube containing 700 µL of buffer A (see Table 1) and incubate on ice for 30 min, protected from light.
   2. Disrupt the tissue for 30 s or until completely homogenized with a hand-held tissue homogenizer. Place the samples on ice and allow the foam to subside for another 10 min.
      NOTE: An aluminum baking sheet placed on dry ice provides a stable working surface and platform for the initial processing/mincing of the tissue.
2. Alternatively, use adherent cell cultures in 100 mm culture dishes as the starting material.
   1. Keep the cells on ice and use a serological pipette to rinse the cells twice with 10 mL of ice-cold PBS containing 100 mM NEM.
   2. Lyse the cells by adding 1 mL of cold homogenization/lysis buffer and scraping vigorously with a rigid cell scraper. Transfer the lysate to a 2 mL centrifuge tube using a micropipette.
      NOTE: Rinsing buffer and lysis buffer may be scaled accordingly to different size culture vessels. Typically, 2-5 million cells are required; however, this varies depending on the lysis efficiency and protein yield for specific cell types. Homogenization buffer may be prepared without NEM for samples being analyzed for total thiols.
3. Transfer the resulting homogenate (step 1.1.2 or 1.2.2) to a 2 mL centrifuge tube using a micropipette, and centrifuge at full speed (≥16,000 × g) at 4 °C for 10 min.
4. Transfer the supernatant (~700 µL or ~1 mL for cell culture) using a micropipette to a 5 mL conical microcentrifuge tube and incubate for 30 min at 55 °C in the dark with shaking at 850 rpm.
5. Using a glass serological pipette, add 4 mL of ice-cold acetone to the samples and incubate at -20 °C overnight for precipitation of protein and removal of excess N-ethylmaleimide.

2. Resin-assisted capture

1. Wash the precipitated protein pellets twice with acetone by centrifuging at 20,500 × g at 4 °C for 10 min, decanting the acetone, removing any remaining acetone using a micropipette, and adding 3 mL of fresh, ice-cold acetone using a glass serological pipette. Invert several times to mix. After the second wash, allow the pellets to air-dry for 1-2 min, being careful not to over-dry as resuspension may become difficult.
2. Using a micropipette, add 1 mL of buffer B (see Table 1) and solubilize the protein using repeated sonication for 15-30 s at a time using a bath sonicator with an output of 250 W and brief vortexing. Measure the protein concentration using the bicinchoninic acid (BCA) assay according to the manufacturer's protocol.
3. To standardize the protein concentrations across samples for further processing and ensure complete removal of NEM, transfer 500 µg of protein to a 0.5 mL 10 kDa centrifugal filter using a micropipette and adjust to a final volume of 500 µL with resuspension buffer.
4. Centrifuge at 14,000 × g at room temperature until the volume in the centrifugal filter is less than 100 µL. Collect the samples by inverting the filter in a collection tube. Centrifuge at 1,000 × g for 2 min and adjust to a final volume of 500 µL using buffer C (see Table 1).
5. Reduce the protein thiols by adding 20 µL of 500 mM dithiothreitol (DTT) using a micropipette to a final concentration of 20 mM and incubating the samples for 30 min at 37 °C while shaking at 850 rpm.
6. After reduction, transfer the samples using a micropipette to 0.5 mL 10 kDa centrifugal filters and centrifuge for 15 min at 14,000 × g at room temperature or until the volume in the centrifugal filter is less than 100 µL. Add buffer D (see Table 1) to make up the volume in the centrifugal filter to 500 µL.
   1. Repeat the centrifuging and addition to 500 µL in step 2.6 three times, and after the fourth centrifugation, collect the samples by inverting the filter in a collection tube and centrifuging at 1,000 × g for 2 min.
7. Measure the protein concentration using the BCA assay according to the manufacturer's protocol.
8. During this buffer exchange, prepare the thiol-affinity resin by weighing the appropriate amount of resin (30 mg/sample) using a microbalance and transferring it to a 50 mL centrifuge tube. Then, using a serological pipette, add water for a final concentration of 30 mg/mL resin and incubate at room temperature for 1 h with agitation for proper hydration of the resin.
   NOTE: The thiol-affinity resin mentioned above has been discontinued by the manufacturer. A possible replacement for this thiol-affinity resin is commercially available. However, this replacement has a nearly 5-fold less binding capacity (see Supplemental Information). Alternatively, the thiol-affinity resin can be synthesized using 2-(pyridyldithio) ethylamine hydrochloride and N-hydroxysuccinimide-activated resin (see Supplemental Information).
   1. After hydration of the resin, place the spin columns on a vacuum manifold and transfer 500 µL of the resin slurry using a micropipette to each column. Apply vacuum for removal of water; repeat this step once to obtain a total of 30 mg of resin per column. Alternatively, centrifuge at 1,000 × g for 2 min instead of using the vacuum manifold for this and all the resin washing and elution steps.
      NOTE: Cutting the end of a 1000 µL pipette tip to increase the bore size helps with the transfer of the resin. It is important to triturate between pipetting to ensure that the resin remains suspended and homogeneous and equal amounts of resin are transferred to each column.
   2. Wash the resin by adding 500 µL of ultrapure water with a micropipette and applying vacuum for removal of the water; repeat this 5 times. Then, wash the resin 5 times with 500 µL of buffer E (see Table 1).
      NOTE: Alternatively, centrifugation at 1,000 x g for 2 min may be used in place of a vacuum manifold for all subsequent wash steps. All the proceeding wash steps are performed with a volume of 500 µL. When adding wash buffers to the column, carefully add with enough force to fully resuspend the resin while avoiding splashing and loss of resin; this allows for complete and efficient washing of resin.
9. Using a micropipette, transfer 150 µg of protein from each reduced sample to a new tube and adjust to a final volume of 120 µL of buffer C (see Table 1). Transfer the protein solution using a micropipette to a plugged spin column containing the resin, place the cap on the column, and incubate for 2 h at room temperature with shaking at 850 rpm.
10. Wash the resin five times with 25 mM HEPES, pH 7.0; 8 M urea; followed by five times with 2 M NaCl; followed by five times with 80% acetonitrile (ACN) with 0.1% trifluoroacetic acid (TFA); and finally five times with 25 mM HEPES, pH 7.7, as described in step 2.8.2 and replace the plug.
    NOTE: Samples may be eluted here for analysis at the protein level (e.g., SDS-polyacrylamide gel electrophoresis (SDS-PAGE), western blot) as described in step 4.1.

3. On-resin tryptic digestion and TMT labeling

1. Prepare enough sequencing-grade modified trypsin solution for 6-8 µg per sample by solubilizing it at a concentration of 0.5 µg/µL in buffer C (see Table 1) so that the final volume allows for at least 120 µL per sample. Using a micropipette, add 120 µL of this trypsin solution to the samples and incubate overnight at 37 °C with shaking at 850 rpm.
   NOTE: To increase the digestion efficiency, an additional digestion step can be included the next day by removing the trypsin solution and replacing it with fresh solution, and continue the digestion for 2 h.
2. Wash the resin five times with 25 mM HEPES, pH 7.0; followed by five times 2 M NaCl; followed by five times with 80% ACN with 0.1% TFA; followed by three times with 25 mM HEPES, pH 7.7. Finally, wash the resin two times with 50 mM triethyl ammonium bicarbonate buffer (TEAB) and replace the plug.
3. Prepare TMT labeling reagents by first allowing them to warm to room temperature before spinning down briefly using a centrifuge at 16,000 × g. Add 150 µL of anhydrous ACN to each vial of TMT labeling reagent using a micropipette. Incubate the vials at room temperature on a thermomixer set to 850 rpm for 5 min to solubilize the reagent completely. Briefly vortex and spin down at 16,000 × g to collect the reagent.
4. Using a micropipette, add 40 µL of 100 mM TEAB to the washed resin, then add 70 µL of the dissolved TMT reagent and incubate for 1 h at room temperature with shaking at 850 rpm. Store any remaining TMT reagent at -80 °C.
   NOTE: Take note of the individual TMT labels assigned to each biological sample (Figure 1).
5. Wash the resin five times with 80% ACN with 0.1% TFA, three times with 100 mM ammonium bicarbonate buffer (ABC), pH 8.0, and twice with water as previously described and replace the plug.

4. Peptide elution

1. Elute the labeled peptides by adding 100 µL of 20 mM DTT in 100 mM ABC, pH 8.0, to each column using a micropipette and incubate at room temperature for 30 min on a thermomixer set to 850 rpm.
   NOTE: After the addition of DTT, the resin will clump. The resin can be disrupted with a pipette tip to break up clumps and ensure the complete elution of the peptides.
2. After this incubation, place the column on a vacuum manifold intended for solid-phase extraction (SPE), apply vacuum, and elute the samples into a 5 mL microcentrifuge tube. Repeat this step once.
3. Finally, add 100 µL of 80% ACN with 0.1% TFA, incubate for 10 min at room temperature, and elute into the same 5 mL centrifuge tube. Collect all the fractions in the same 5 mL microcentrifuge tube.
   NOTE: To prevent sample loss, low binding tubes must be used for elution, and volumes must be kept at or below a volume of 4.0 mL for a single 5 mL tube.
4. Place the eluted samples in a vacuum concentrator until dry. Store the dry peptides at -80 °C and resuspend them later.
   ​NOTE: Samples may also be eluted separately, and an aliquot may be removed and analyzed by SDS-PAGE for analysis at the peptide level before combining the samples.

5. Peptide alkylation and desalting/clean-up

1. Resuspend the dried peptides by adding a small volume of 100 mM ABC buffer, pH 8.0 (no greater than 500 µL), using a micropipette. Use repeated sonication for 15-30 s at a time using a bath sonicator with an output of 250 W and vortex to solubilize and transfer to a 2 mL tube.
   NOTE: The volume of 100 mM ABC, pH 8.0 to be added is based on the volume needed to resuspend the DTT at a molarity of 150 mM. Users will need to determine the amount of DTT present in their sample based on what was added originally in step 4.1.
2. Add enough concentrated stock solution (600 mM) of iodoacetamide (IAA) dissolved in ABC using a micropipette to achieve a 1:4 molar ratio of DTT:IAA and incubate the samples at RT for 1 h with shaking at 850 rpm.
3. Acidify the samples to pH < 3 by adding concentrated TFA (10%) using a micropipette and perform sample desalting using reverse-phase clean-up according to the manufacturer's instructions.
4. Place the clean peptides in a vacuum concentrator until dry. Store the dry peptides at -80 °C until further analysis.

6. Liquid chromatography-tandem mass spectrometry

1. Resuspend the dried peptides by repeated sonication for 15-30 s at a time using a bath sonicator with an output of 250 W and vortexing in 20-40 µL of water containing 3% ACN. Determine the peptide concentration by performing a BCA assay according to the manufacturer's protocol.
2. Separate the samples by reversed-phase LC and MS/MS as previously described⁶ and record the MS1 spectra over the m/z range of 400-2000. Ensure high-energy collisional dissociation (HCD) is utilized to obtain reporter ion intensity information for the analysis of isobarically labeled peptides. See the methods sections of previous reports for more details about instrument run conditions^27,30 and the analysis of MS data^27,31.
   NOTE: Different LC-MS/MS systems or settings can be used to analyze the peptide samples. The coverage and sensitivity of peptide identification will depend on the particular system and settings used.