Tandem affinity purification and mass spectrometry

Monobodies cloned into the retroviral TAP tagged vector were used to establish Jurkat and K562 stable cell lines. Cells expressing the IRES-GFP were selected and sorted using FACS. The TAP purification consisted in the lysis of used 2–3 × 10⁹ cells as previously described⁵⁸. Briefly, the elution of the protein complexes following two steps of affinity purification was performed using 0.1 M hydrochloric acid immediately followed by the neutralization using 0.5 M Triethyl ammonium bicarbonate. Samples were then boiled and 10% of the eluates were resolved by SDS-PAGE (4–20% gel; Bio-rad) and silver stained to assess the efficiency of the pull-down. The rest of the eluates were then separated by SDS-PAGE and stained with R-250 Coomassie Blue Solution. For all single biological experiments, the entire SDS-PAGE gel lanes were first sliced into 4 fractions resulting in a total of 16 samples, which were washed twice in 50% ethanol and 50 mM ammonium bicarbonate (AB, Sigma-Aldrich) for 20 min and dried by vacuum centrifugation. Sample reduction was performed with 10 mM dithioerythritol (Merck-Millipore) for 1 h at 56 °C. A washing-drying step as described above was repeated before the alkylation step performed with 55 mM Iodoacetamide (Sigma-Aldrich) for 45 min at 37 °C in the dark. Samples were washed-dried again and digested overnight at 37 °C using modified mass spectrometry grade trypsin (Trypsin Gold, Promega) at a concentration of 12.5 ng µl⁻¹ in 50 mM AB and 10 mM CaCl₂. Resulting peptides were extracted in 70% ethanol, 5% formic acid (FA, Merck-Millipore) twice for 20 min with permanent shaking. Samples were further dried by vacuum centrifugation and stored at −20 °C. Peptides were desalted on StageTips⁵⁹ and dried under a vacuum concentrator prior to LC-MS/MS injection. Samples were resuspended in 2% acetonitrile (Biosolve), 0.1% FA and nano-flow separations were performed on a Dionex Ultimate 3000 RSLC nano UPLC system (Thermo Fischer Scientific) on-line connected with a Q Exactive Orbitrap Mass Spectrometer (Thermo Fischer Scientific). Acquisitions were performed using Data-Dependent acquisition mode. First MS scans were acquired with a resolution of 70,000 (at 200 m/z) and the 12 most intense parent ions were selected and fragmented by High energy Collision Dissociation (HCD) with a Normalized Collision Energy (NCE) of 27% using an isolation window of 1.2 m/z. Fragmented ions were acquired with a resolution 17’500 (at 200 m/z) and then excluded for the following 30 s. A capillary precolumn (Acclaim Pepmap C18, 3 μm-100 Å, 2 cm × 75 μm ID) was used for sample trapping and cleaning. A 50 cm long capillary column (75 μm ID; in-house packed using ReproSil-Pur C18-AQ 1.9 μm silica beads; Dr. Maisch) was then used for analytical separations at 250 nl min⁻¹ over 150 min. biphasic gradients. Raw data were processed using SEQUEST (precursor and fragment ion mass tolerances were 10 ppm and 0.05 Da, respectively) in Proteome Discoverer v.2.2 against a concatenated database consisting of the Uniprot human protein database (95796 entries) and monobodies sequences. Enzyme specificity was set to Trypsin and a minimum of six amino acids was required for peptide identification. Up to two missed cleavages were allowed. A cut-off was fixed at 1% FDR at the peptide and protein identification level. For the interactome database search carbamidomethylation was set as a fixed modification, whereas oxidation (M), acetylation (protein N-term), PyroGlu (N-term Q), were considered as variable modifications. Data was further processed and inspected in Scaffold 4.10 (Proteome Software, Portland, USA) and spectra of interest were manually validated. In order to analyze data in an unbiased way and filter for most unspecific protein contaminants, a selectivity score was calculated as a function of the number of TAP experiments similarly performed in Jurkat and K562 cells using unrelated monobodies (including the HA4-Y87A control)^35,37,44,60 a given protein was detected in, as compared to the total number of TAP experiments we have performed and previously published. A threshold of >75% was applied to determine the likelihood of a protein being a specific monobody interactor. This scoring permits the identification of proteins found in only <25% of all the TAPs in our internal database, thus allowing most unspecific contaminating or highly expressed cytosolic proteins recovered from the pull down to be filtered out. Common contaminants and unspecific proteins identified were included in protein ranking shown in Table 2 for transparency. All mass spectrometry proteomics data from TAPs have been deposited to the ProteomeXchange Consortium via the PRIDE⁶¹ partner repository with the dataset identifier PXD018374.