Cellular Depletion of Major Cathepsin Proteases in HeLa and SH-SY5Y Cells

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HeLa WT and CTS DBLZ-deficient as well as SH-SY5Y WT and CTS DBL-deficient cell clones (each n=6) were seeded 24 h after cell pellet collection. Cells were washed in ice cold PBS and harvested with a cell scraper. A modified RIPA lysis buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 50 mM TrisHCl pH 8) was used to lyse the cells one ice for 30 min. The supernatant was collected after centrifugation at 16.000 rpm for 10 min at 4°C. 10 μl were used to determine protein concentration using a BCA assay. Per sample 25 μg protein was subjected to the filter-aided sample preparation protocol (FASP) using Vivacon spin filters with a 30 kDa cut-off (Sartorius). Proteins were reduced with 20 mM DTT followed by alkylation of cysteine residues with 50 mM iodoacetamide (Sigma Aldrich). Next, proteins were washed with 8 M urea (Sigma Aldrich) and digested first with 0.5 μg LysC (Promega) for 16 h at 37 °C and second with 0.25 μg trypsin (Promega) for 4 h at 37 °C. Generated peptides were eluted by centrifugation and acidified with 8 % (v/v) formic acid to pH<3. Proteolytic peptides were desalted by stop-and-go extraction (STAGE) with self-packed C18 tips (Empore) and dried by vacuum centrifugation [57]. Peptides were dissolved in 20 μl 0.1 % formic acid (FA) and protein concentration was determined by a nanodrop photometer at 280 nm. Peptides were analyzed for label-free protein quantification (LFQ) on an Easy nLC 1200 nanoHPLC (Thermo Scientific) which was coupled online to a Nanospray Flex Ion Source (Thermo Sientific) equipped with a PRSO-V1 column oven (Sonation) to a Q-Exactive HF mass spectrometer (Thermo Scientific). 1.3 μg of peptides were separated on in-house packed C18 columns (30 cm x 75 μm ID, ReproSil-Pur 120 C18-AQ, 1.9 μm, Dr. Maisch GmbH) using a binary gradient of water (A) and acetonitrile (B) supplemented with 0.1% formic acid (0 min, 2% B; 3:30 min, 5% B; 137:30 min, 25% B; 168:30 min, 35% B; 182:30 min, 60% B) at 50°C column temperature. Data-dependent acquisition (DDA) was used for LFQ. Full MS scans were acquired at a resolution of 120,000 (m/z range: 300-1400; automatic gain control (AGC) target: 3E+6). The 15 most intense peptide ions per full MS scan were selected for peptide fragmentation (resolution: 15,000; isolation width: 1.6 m/z; AGC target: 1E+5; normalized collision energy (NCE): 26%). A dynamic exclusion of 120 s was used for peptide fragmentation.