Ge 130 ultrasonic processor

E. coli (K12) was grown to stationary phase in LB media. E. coli cells were pelleted at 1500g for 10 minutes and washed with phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄) followed by cross-linking buffer (180 mM Sodium phosphate pH 8.0). The cell pellet was gently resuspended in 500 μL cross-linking buffer, and biotin aspartate proline-N-hydroxyphtalamide (BDP-NHP), synthesized by solid phase synthesis^{24 (link)}, was added to a final concentration of 10 mM. After one hour at room-temperature any remaining reactive cross-linker was quenched with the addition of 1mL of 100 mM ammonium bicarbonate. After quenching, the cells were again pelleted, the cross-linking buffer was removed, and the cells were resuspended in 100 mM ammonium bicarbonate. Urea was added to 8M and then the cells were lysed by sonication using a GE-130 ultrasonic processor. The lysed samples were reduced with 5 mM Tris(2-carboxyethyl)phosphine for 30 minutes at room temperature, and then alkylated in 10 mM Iodoacetamide for 45 minutes in the dark. The samples were diluted 10-fold with 100 mM ammonium bicarbonate to reduce urea concentration to 0.8M, and the proteins were then digested overnight at 37°C using trypsin (Promega). Digested samples were desalted using C18 sep-pak columns (Waters).

Frozen fractions were transferred to a stainless-steel cryogrinding jar cooled to −196°C with liquid nitrogen in 0.1M NH₄HCO₃. The samples were cryoground for five 3 min cycles at 30 Hz using a Retch MM 400 mixer mill. Between cycles, the cryogrinder was cooled with liquid nitrogen. The resulting frozen powder was transferred to a falcon tube where 8M urea (in 0.1M Tris, pH 8.0) was added. Samples were sonicated using a GE-130 ultrasonic processor, followed by reduction of cysteine residues by incubation with 5mM Tris (2-carboxyethyl) phosphine (TCEP, Fisher Scientific) for 30 min, followed by alkylation with 45 min incubation with 10mM iodoacetamide (Fisher Scientific). Urea concentration was reduced to less than 1M by diluting the samples by a factor of 10 with fresh 0.1M Tris buffer (pH 8.0). The protein concentration was measured using the Pierce Coomassie protein assay (Thermo Scientific).