Dithiothreitol (dtt)

ZnCl₂, FeCl₃, Na₂S nonahydrate, ampicillin sodium salt, KCl, Polycytidylic acid, Bovine Serum Albumin, ZnSO₄, urea, trifluoracetic acid (TFA) (HPLC grade), Methanol (HPLC grade), SP sepharose resin, glycerol, and RNA (HPLC purified grade) were obtained from Sigma-Aldrich. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was purchased from Research Products International. BL21-DE3 competent cells and amylose resin was acquired from New England Biolabs. Luria Bertani Lennox Broth (LB) and dithiothreitol (DTT) were obtained from American Bio. Glucose was purchased from Calbiochem. Tris base (2-amino-2-(hydroxymethyl)propane-1,3-diol), NaCl, nitric acid (trace metal grade), acetonitrile (HPLC grade), and hydrochloric acid (trace metal grade) were acquired from Fisher Scientific. Iron, zinc, germanium, and scandium ICP-MS standards were obtained from Fluka analytical. Bismuth ICP-MS standard was purchased from Ricca Chemical Company. Rhodium ICP-MS standard was acquired from VWR analytical. ICP-MS tuning solution was obtained from Agilent. MES was acquired from Amresco. TCEP and protease and phosphatase inhibitor tablets were obtained from Thermo Scientific. 4,4’-dithiodipyridine (DTDP) was purchased from Acros Organics. CoCl₂ was acquired from Merck.

Thirty milliliters of HEK293-6E cells (National Research Council of Canada, Ottawa, Canada) at 1 × 10⁶ cells/ml were pelleted by centrifugation and washed with 5 ml chilled 1x PBS. Each pellet was resuspended in 300 μl lysis buffer (RIPA buffer [MilliporeSigma] containing 3.2 mM sodium orthovanadate [NEB] and 10 μg each leupeptin, pepstatin, aprotinin and chymostatin [MilliporeSigma]), and incubated on ice for 15 min. Pellets were then combined and sonicated three times for 2 s each time and kept on ice. Resulting lysates were centrifuged for 10 min at 8000×g and quantified at A280 using a microplate reader. Lysates were then mixed with equal volume of 2x Laemmli Sample Buffer (Bio-Rad Laboratories) containing 200 mM dithiothreitol (AmericanBio) and denatured at 95°C for 10 min.

The following components (per reaction) were mixed in a 96-well deep-well plate: 0.145 mg purified modMBP, 10x Sortase buffer [200 mM Tris-HCl, 1.5 M NaCl, 50 mM CaCl₂, 2 mM betamercaptoethanol], 0.04-mg target peptide (design described above), 3-μg sortase enzyme [Moradec, CA, USA] and 1x phosphate-buffered saline (PBS) to final volume of 113 μl. One well without target peptide was used as a negative ‘unconjugated’ control. The plate was incubated with shaking at 250 rpm at 37°C for 2 h. Reactions were then mixed with equal volume of 2x Laemmli Sample Buffer (Bio-Rad Laboratories, CA, USA) containing 200 mM dithiothreitol (AmericanBio) and denatured at 95°C for 10 min.