Chymotrypsin-digested sample of one independent experiment of chromosomal hipA7 (Rep1) was fractionated using an offline peptide fractionation at high pH to increase sequence coverage and detect unmodified GltX peptide. Peptides were loaded onto a reversed-phase XBridge BEH130 C18 3.5 μm 4.6 × 250 mm column installed in an UltiMate 3000 HPLC and detected by ultraviolet at λ = 214 nm at 25°C. The system was operated under basic conditions using buffer A (5 mM NH4OH) and buffer B (5 mM NH4OH in 90% acetonitrile) at pH 10. Peptides were eluted using an 80-min gradient at a flow rate of 1 ml/min. The organic portion was ramped from 5 to 25% B in 45 min to 40% in 10 min and finally to 70% in 5 min, followed by column equilibrated. Fractions were collected in 1-min intervals for 60 min and concatenated evenly into 30 pools. Acetonitrile was evaporated by vacuum centrifugation, and samples were acidified to pH 2 and measured by LC-MS/MS.

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