Development of a proteomic workflow for the identification of heparan sulfate proteoglycan-binding substrates of ADAM17

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Wild-type (WT) and ADAM17 knockout (KO) mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), containing 1% L-Glutamine, 1% Penicilin and Streptomycin, 1% Sodium Pyruvate and 10% Fetal Bovine Serum (FBS) at 37 °C, 5% CO2 (all reagents were purchased from Euroclone, Pero, Italy). HTB94 were kindly provided by Prof Hideaki Nagase, and cultured in DMEM containing 1% L-Glutamine, 1% Penicillin and Streptomycin, 1% Sodium Pyruvate and 10% FBS at 37° C, 5% CO2. WT MEFs were seeded in T175 flasks and grown in complete DMEM until confluence. Cells were washed twice with PBS, incubated with 20 mL serum free DMEM for 24 h. Then, conditioned medium was harvested and aliquoted. 1 mL conditioned media were treated with 200 µg/mL heparin (heparin sodium salt - H3393 - from Sigma-Aldrich) or equal volumes of PBS (untreated controls - CT) for 20 min at RT under agitation. After centrifugation to remove cell debris (7000 x g, 10 min), heparin-treated or untreated conditioned media were subjected to filter aided sample preparation (FASP) with 10 kDa Vivacon 500 spin filters (Sartorius, Göttingen, Germany), as previously described (1). Briefly, samples were reduced and denatured by incubation with 50 mM DTT in UA buffer (8 M urea, 100 mM Tris/HCl, pH 8.5) for 30 minutes. Subsequently, samples were centrifuged to remove excess DTT (14000 x g, 20 min) and alkylated with 50 mM iodoacetamide (IAA) in UA buffer for 5 minutes, at 37 °C in the dark. After reduction and alkylation, samples were washed twice with UB buffer (8 M urea, 100 mM Tris/HCl, pH 8) and digested with 0.2 µg LysC (Promega, Madison, Wisconsin, US) in UC buffer (2 M urea, 25 mM Tris/HCl, pH 8) overnight at 37 °C. Then, samples were subjected to another digestion step with 0.1 µg trypsin (Promega) for 4 hours at 37 °C in 50 mM ammonium bicarbonate. Peptides were eluted in 120 µl of 0.5 M NaCl by subjecting the filter columns to centrifugation (14000 x g for 1 h), and acidified with 20 µl of 8% formic acid. The buffer of the eluted peptides was exchanged by stop-and-go extraction (STAGE) on reverse phase tips packed with C18 disks in-house (Empore SPE Disks, Sigma-Aldrich), as previously described (2). Briefly, after activation with 100 µL methanol, the C18 resin was washed four times with 0.1% formic acid (FA). Peptides were loaded onto the C18-packed STAGE-tips, washed with 0.1% FA and finally eluted with 40 µL of 60% acetonitrile and 0.1% FA in mass spectrometry grade water (all reagents from Sigma-Aldrich). Peptides were then dried by vacuum centrifugation and resuspended in 20 µL of 0.1% FA. 1 µg of peptide mixture per each sample was loaded onto an Easy nLC 1200 system, which was coupled online via a Nanospray Flex Ion Source to a Q-Exactive HF mass spectrometer (all instruments from Thermo Scientific). Peptides were separated on a self-packed C18 column (30 cm × 75 µm ID, 1.9 µm ReproSil-Pur C18-AQ 120 Dr. Maisch GmbH) with 250 nL/min flow using a binary gradient of water (A) and acetonitrile (B) supplemented with 0.1% formic acid for 120 minutes. Data-dependent acquisition (DDA) was used for label free quantification (LFQ). Full mass spectrometry (MS) scans were acquired at a resolution of 120,000 (m/z range: 300–1400; automatic gain control (AGC) target: 3xE6; max injection time 50 ms). The DDA was used on 15 most intense peptide ions per full MS scan for peptide fragmentation (resolution: 15,000; isolation width: 1.6 m/z; AGC target: 1xE5; normalized collision energy (NCE): 26%, max injection time: 100 ms). A dynamic exclusion of 120 s was used for peptide fragmentation. (1) Wisniewski, J. R., Zougman, A., Nagaraj, N., and Mann, M. (2009) Universal sample preparation method for proteome analysis. Nat Methods 6, 359-362 (2) Rappsilber, J., Ishihama, Y., and Mann, M. (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75, 663-670