Xb c8 column

VEGFR1D2 of 20 μm was incubated in phosphate buffer 20 mm and TCEP 5 or 10 mm, pH = 7. All the reactions were monitored by LC‐MS, analyzing protein samples on an Agilent 1200 Infinity Series (Agilent Technologies) equipped with an ESI source and a ToF detector. Protein samples were loaded on an Aeris widepore (150 × 2.1 mm, 3.6 μm) XB‐C8 column (Phenomenex), applying a method with a flow rate of 0.2 mL·min⁻¹ and a linear gradient of CH₃CN (0.05% TFA) in H₂O (0.05% TFA) from 5% to 70% in 20 min. The protein sample precipitated after 24 h of incubation with 10 mm TCEP was centrifuged, the pellet was resuspended in 50 μL of 8 M guanidinium hydrochloride and 1.5 μL were also analyzed.

In order to estimate the oxidation state of the disulfide bridge at 353 K, VEGFR1D2 was diluted up to 10 µm in Tris‐HCl 50 mm, pH = 7.0, containing NaCl 150 mm and incubated for 40 min in a thermostated bath set to the above‐mentioned temperature. Then, iodoacetamide (Sigma‐Aldrich, St. Louis, MO, USA), dissolved at a concentration of 500 mm in the same buffer, was added to the heat‐treated protein domain at a final concentration of 14 mm. The alkylation reaction was allowed to proceed at room temperature, for 30 min, in the dark. As positive control, VEGFR1D2 was first reduced with 5 mm DTT at 353 K for 40 min and then alkylated by iodoacetamide as described for the unreduced sample. Analysis of protein samples was assessed by LC‐MS on an Agilent 1200 Infinity Series (Agilent Technologies) equipped with an ESI source and a ToF detector, using an Aeris widepore (150 × 2.1 mm, 3.6 μm) XB‐C8 column (Phenomenex, Bologna, Italy), applying a method with a flow rate of 0.2 mL·min⁻¹ and a linear gradient of CH₃CN (0.05% TFA) in H₂O (0.05% TFA) from 5% to 70% in 20 min.