TRP14 is the rate limiting enzyme for intracellular cystine reduction and regulates proteome cysteinylation. Figure 2C
Identification of cysteinylated proteins in parental HEK cells and TRP14 knockdown HEK cells. For the obtention of cysteinylated cell lysates, both control and TRP14 KD cell cultures were incubated with 250 µM biotinylated cysteine (Cys-BIO) for 1 h, the incubation medium was discarded, cells were washed with ice-cold PBS and collected in 300 µL of lysis buffer containing 50 mM Tris-HCl (pH 7.5), 2 mM EDTA, 150 mM NaCl, 1% Triton, 10 mM N-ethylmaleimide (NEM), and a protease inhibitor cocktail (Sigma-Aldrich, Missouri, USA). Cells were lysed by three freezing-thawing cycles, and cell lysates were centrifuged at 3500 rpm for 10 min to collect supernatants for analysis. Pierce™ Monomeric Avidin Agarose Kit (Thermo Scientific, Massachusetts, USA) was used to purify cysteinylated proteins tagged with biotin according to the manufacturer’s instructions. 14 µg were loaded in a 1D PAGE gel , which were later in-gel reduced with dithiothreitol (DTT) and S-alkylated with iodoacetamide as previously described50. Samples were digested with 100 ng sequencing-grade trypsin (Promega, Madison, USA). The digestion mixture was dried and re-suspended in 20 µL of 2% acetonitrile, and 0.1% trifluoroacetic acid (TFA). 5 µL of sample were loaded onto a trap column (NanoLC Column, 3µ C18-CL, 350 µm x 0.5 mm; Eksigent) and desalted with 0.1% TFA at 2 µL/min during 10 min. Desalted samples were then loaded into an analytical column (LC Column, 3C18-CL-120, 3 µ, 120 Å 75 µm x 15 cm, Eksigent) equilibrated with 5% acetonitrile 0.1% formic acid. Elution was carried out with a linear gradient of 5 to 35% B in A for 60 min (A: 0.1% formic acid; B: acetonitrile, 0.1% formic acid) at a flow rate of 300 nL/min. Eluted samples were analyzed in a mass spectrometer (5600 TripleTOF, ABSCIEX, Massachusetts, USA). Samples were ionized applying 2.8 kV to the spray emitter. Analysis was carried out in a data-dependent mode. Survey MS1 scans were acquired from 350-1250 m/z for 250 ms. The quadrupole resolution was set to ‘UNIT’ for MS2 experiments, which were acquired at 100-1500 m/z for 50 ms in the high sensitivity mode. Up to 25 ions were selected for fragmentation after each survey scan. Dynamic exclusion was set to 15 s. ProteinPilot default parameters were used to generate a peak list from 5600 TripleTof wiff files. The Paragon algorithm was used to search the SwisProt database with the parameters: trypsin specificity and cys-alkylation with taxonomy restricted to human, and false discovery rate (FDR) correction for proteins. All eluted samples from all experiments were combined to create a spectra library for differential expression analysis. Peak View 1.1. software (Sciex, Massachusetts, USA) was used to quantify the areas for all the peptides assigned in the library and the computed areas for proteins.