Vivaspin column

Overnight cultures of E. coli strain BL21 (DE3) harboring the different pET41b-OXA-48/variant/mutated were used to inoculate 2 L of LB broth containing 50 µg/mL of kanamycin. Bacteria were cultured at 37 °C until reaching an OD of 0.6 at 600 nm. Expression of the different OXA-48/variant/mutations were induced overnight at 25 °C with 0.2 mM IPTG, as previously described [14 (link)]. OXA-48-like enzymes were purified in one-step pseudo-affinity chromatography using an NTA–nickel column (GE Healthcare, Les Ulis, France), as previously described [14 (link)]. Protein purity was estimated by SDS–PAGE, pure fractions were pooled and dialyzed against 20 mM Hepes, 50 mM K₂SO₄ buffer (pH 7.0) and concentrated by using Vivaspin^® columns (GE Healthcare, Les Ulis, France). Protein concentration was determined by Bradford Protein assay (Bio-Rad, Marnes-La-Coquette, France) [23 (link)].

Recombinant ALK intracellular domain (ICD, amino acids 1064-1620) was expressed in Sf9 cells as a cleavable GST-tagged product using the BacPAK Baculovirus expression system (Clontech). Cells were infected at a MOI = 5 and harvested after 72 hours, as described [24 (link)] and lysed in lysis buffer (50mM Tris-HCl, pH 8.0, 100mM NaCl, 1mM DTT, 0.5mM EDTA, 0.1% Triton-X100, and protease inhibitors: leupeptin, aprotinin, benzamidine, pepstatin A). The total lysate was loaded onto Glutathione Sepharose 4B resin (GE Healthcare) and incubated for 2 hours at 4°C with rotation. After extensive washing, the rALK-ICD was eluted by on-column overnight cleavage by GST-3C protease and concentrated using VivaSpin columns (GE Healthcare, cut-off 10kDa) to a final concentration of 0.3 mg/ml. ALK peptides were synthetized and purified by C S Bio Co., Menlo Park, CA. The full sequence of peptides is presented in Supplementary Table 1. Each peptide was 36 amino acids long, with a 12-amino acid overlap between flanking peptides.

Site-specific N-glycosylation analysis was performed using proteolytic digestion followed by tandem LC-MS. Prior to digestion, trimers were denatured, reduced and alkylated by incubation for 1h at room temperature (RT) in a 50 mM Tris/HCl, pH 8.0 buffer containing 6 M urea and 5 mM dithiothreitol (DTT), followed by the addition of 20 mM iodacetamide (IAA) for a further 1h at RT in the dark, and then additional DTT (20 mM) for another 1h, to eliminate any residual IAA. The alkylated trimers were buffer-exchanged into 50 mM Tris/HCl, pH 8.0 using Vivaspin columns (GE healthcare) and digested separately with trypsin, elastase and chymotrypsin (Mass Spectrometry Grade, Promega) at a ratio of 1:30 (w/w). Glycopeptides were selected from the protease-digested samples using the ProteoExtract Glycopeptide Enrichment Kit (Merck Millipore) following the manufacturer’s protocol. Enriched glycopeptides were analyzed by LC-ESI MS on an Orbitrap fusion mass spectrometer (Thermo Fisher Scientific), as previously described (Behrens et al., 2016 (link)), using higher energy collisional dissociation (HCD) fragmentation. Data analysis and glycopeptide identification were performed using ByonicTM (Version 2.7) and ByologicTM software (Version 2.3; Protein Metrics Inc.), as previously described (Behrens et al., 2016 (link)).