6300 series trap control version 6

The HPLC-MS-system: Agilent HPLC-MS-system consisting of: 1200 binary pump, 1200 automatic sampler, thermostated, 1200 column thermostate, 1200 diode array detector with 10 mm standard flowcell, LC/MSD Ultra Trap System XCT 6330 (Agilent, Waldbronn).
HPLC parameters: stationary phase: Nucleosil 100 C18 3 µm, 100 × 2 mm ID with a precolumn 10 × 2 mm ID (Dr. Maisch GmbH, Ammerbuch), column temperature: 40 °C, mobile Phase: A = 0.1% formic acid, B = 0.06% formic acid in acetonitrile, gradient: t₀ = 10% B, t₁₅ = t₁₇ = 100% B, posttime 6 min. 10% B, flowrate: 400 µL/min, injection volume: 2.5 µL, detections wavelengths (bandwidth): 230 nm (10 nm), 260 nm (20 nm), 280 nm (20 nm), 360 nm (20 nm), 435 nm (40 nm), software: LC/MSD ChemStation Rev. B.01.03, Agilent.
MS parameters: ionisation: ESI (positive and negative, alternating), mode: ultra-scan, capillary voltage: 3.5 kV, temperature: 350 °C, target mass: m/z 800, software: 6300 series trap control version 6.1, Bruker Daltonik (Agilent, Waldbronn, Germany).

To confirm the stability of 12IGF under standard assay conditions (i.e., to exclude proteolysis of 12IGF by ClpP), HPLC-MS analysis was performed on an Agilent 1200 HPLC series equipped with a temperature-controlled sampler and coupled to an Ultra Trap System XCT 6330 (Agilent, Waldbronn, Germany). 5 μL of sample (1 μM SaClpP, 46 μM 12IGF in SaClpP activity buffer) were injected at given time points into a Reprosil-Gold 300 C-18 5 μm column (10 × 2 mm precolumn and 100 × 2 mm separation column, Dr. Maisch HPLC GmbH, Ammerbuch, Germany). Separation was conducted with a linear gradient elution over 55 min from 0% B to 100% B at a flow rate of 400 μL/min. Eluents A and B were composed of 0.1% formic acid in water and 0.06% formic acid in methanol, respectively. Absorption spectra were recorded at wavelengths 220 nm, 260 nm, 280 nm, 360 nm, and 435 nm, while 12IGF showed the strongest signal at 220 nm. Electronspray ionization (ESI) was performed in ultra-scan mode (positive and negative, alternating) with a capillary voltage of 3.5 kV and a drying gas temperature of 350 °C. Data analysis was performed with the software 6300 Series Trap Control Version 6.1 (Bruker Corporation, Billerica, MA, USA).