2.6. Oxidation Detection (OxICAT)

In the OxICAT method, reduced cysteines are specifically differentially labeled with ¹²C ICAT and oxidized cysteines after their reduction with ¹³C ICAT. The ICAT reagent also contains an affinity tag, which facilitates isolation and concentration of tagged peptides before MS analysis. OxICAT labeling was performed, as follows.

The starting materials for the OxICAT method (Cleavable ICAT^® reagent kit for protein labeling, (Sciex, Framingham, USA), not commercially available at present) consisted of frozen human heart tissue material with a size of approximately 2 × 2 mm stored in liquid nitrogen. The following steps were performed under anaerobic conditions to protect the tissue from disease-unrelated oxidation events. Before starting, the DAB buffer (6 M urea, 10 mM EDTA, 200 mM Tris/HCl with pH 8.5 and 0.5% SDS) was prepared and incubated in an anaerobic environment for 4 h to remove all oxygen from the solution. The tissue samples were placed in the anaerobic chamber, and 80 µL DAB buffer with 20 µL acetonitrile (ACN) were added to homogenize the tissue. For each tissue homogenate, one vial of the light ICAT reagent was used and samples (n = 5 per group) were incubated for 2 h at 37 °C and 1300 rpm in the dark. The next steps were performed under normal aerobic conditions. The reaction was stopped by adding 400 µL of ice-cold acetone and proteins were precipitated over night at −20 °C. The next day, samples were centrifuged at 13,000× g for 30 min at 4 °C. The supernatant was then discarded and another 400 µL of ice-cold acetone was added for washing. After repeating the centrifugation step, the remaining acetone was removed by evaporation at 25 °C until samples were completely dry. The protein pellets were resuspended in 80 µL of DAB buffer. For reducing oxidized cysteins, 2 µL of 50 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to the samples and incubated at 37 °C for 10 min at 300 rpm. Afterwards, 20 µL of ACN was combined with one vial of heavy ICAT. Each sample was filled into one vial and incubated for 2 h at 37 °C and 1.300 rpm. Again, proteins were precipitated via 400 µL of ice-cold acetone for 4 hat −20 °C before being centrifuged and washed twice. After the last washing step, the acetone was evaporated at 25 °C. The dried protein pellets were mixed with 80 µL of denaturing buffer (50 mM Tris pH 8.5 and 0.1% SDS) and 20 µL of ACN. The samples were digested with trypsin over night at 37 °C. The following cation exchange and affinity chromatography were performed according to the manufacturer’s protocol and components of the kit. During these steps, all unlabeled and non-cysteine-containing peptides were removed. For mass spectrometry analysis, the flow-through containing the digested and labeled peptides was dried in a vacuum centrifuge. To remove the biotin tag, the two cleavage reagents A and B (95:5) were mixed and 90 µL was added to each sample. After vortexing, the mixture was incubated at 37 °C for 2 h at 300 rpm. The samples were concentrated in a vacuum centrifuge for 20 min. Then, 4 µL of sample was combined with 46 µL of a 0.1% trifluoroacetic acid (TFA) solution of which 30 µL was filled into HPLC tubes. Afterwards, the peptide samples were analyzed using mass spectrometry.