Bioconfirm software

Digestion of Spy alone (50 µM) or Spy-Im7 L53A I54A complex (50 µM each) was carried out at 1:100 mass ratio of trypsin to protein in 40 mM HEPES, 100 mM NaCl, pH 7.5 at room temperature. At different time points (0–8 min), aliquots were withdrawn and the digestion was stopped with 10% TFA. Peptides were separated by a reverse phase C18 column (Zorbax 300SB-C18, 1 × 50 mm, 3.5 µm, Agilent, Santa Clara, CA) at room temperature and then applied to a Q-TOF dual ESI LC/MS (Agilent) for identification. A 15 min linear gradient of 2–80% acetonitrile in 0.1% formic acid at a flow rate of 0.3 µl/min was used to elute the peptides. Peptide identification was performed using BioConfirm software (Agilent).

First, 1 mg/ml of TVD–Fab before and after conjugation was prepared for mass spectrometry and data were obtained on an Agilent Electrospray Ionization Time of Flight (ESI-TOF) mass spectrometer. Deconvoluted masses were obtained using Agilent BioConfirm Software.

To probe for a reactive oxygen species mediated protein-based modification in the enzymatic assays, LC-MS analysis was performed using an Agilent G6545A qTOF mass spectrometer equipped with a dual AJS ESI source and an Agilent 1290 Infinity series diode array detector, autosampler, and binary pump. For these experiments, an Aeris WIDEPORE C4 column (2.1 × 50 mm, 3.6 µm, 200 Å) (Phenomenex) was employed. Each tested sample contained 10 µM oxygenase, 40 µM reductase, 2 mM substrate, 500 µM NADH, and 100 µM ferrous ammonium sulfate hexahydrate and was incubated for 3 h prior to analysis. The LC-MS solvents used were solvent A (95% water, 5% acetonitrile, and 0.1% formic acid) and solvent B (95% acetonitrile, 5% water, and 0.1% formic acid). The seven-minute chromatographic method used for these experiments employed a gradient that ran from 5% to 95% Solvent B with a flow rate of 0.3 mL/min. Expected protein molecular weights were calculated using Agilent BioConfirm software.

The conjugation site of p-SCN-Bn-DOTA to the C595 mAb was determined using mass spectrometry (MS). Samples of DOTA-C595 conjugate and unmodified C595 were buffer exchanged into 0.2 M ammonium acetate using a 50 kDa Amicon^® centrifugal filter unit and diluted to 3 mg/mL. Samples were reduced into light and heavy antibody chains through 1:10 addition of 0.1 M of dithiothreitol (DTT) prepared in 0.2 M ammonium acetate. The samples were incubated with DTT at 37 °C for up to 60 min before reading analysis by MS. Protein mass measurements were carried out under denaturing conditions using an Agilent 6230 time-of-flight instrument coupled to an Agilent 1260 Infinity II LC System (Agilent Technologies, Santa Clara, CA, USA). Protein sample (5 µL) was injected and electrosprayed using 50% aqueous acetonitrile/0.01% formic acid at a flow rate of 0.2 mL/min, without chromatographic separation. ESI-MS conditions were: positive-ion mode; capillary voltage, 4000 V; nozzle voltage 2000 V; fragmentor, 200 V; gas 13 L/min; gas temperature, 325 °C; sheath gas 11 L/min; and sheath gas temperature, 350 °C. Spectra were deconvoluted using BioConfirm software (Agilent Technologies, Santa Clara, CA, USA).