Biopharmaview v 3

Protein samples were analyzed by LC/MS using a Sciex X500B Q-ToF mass spectrometer running Sciex OS v.1.6.1, coupled to an Agilent 1,290 Infinity II HPLC. Samples were injected onto a POROS R1 reverse-phase column (2.1 × 30 mm, 20 µm particle size, 4000 Å pore size), desalted, and the amount of buffer B was manually increased stepwise until the protein eluted off the column. Buffer A contained 0.1% formic acid in water and buffer B contained 0.1% formic acid in acetonitrile. The mobile phase flow rate was 300 µL/min. The acquired mass spectra for the protein of interest were deconvoluted using BioPharmaView v. 3.0.1 software (Sciex) in order to obtain the molecular weight.

Protein samples were analysed by LC–MS, using a Sciex X500B Q-TOF mass spectrometer coupled to an Agilent 1290 Infinity II HPLC. Samples were injected onto a POROS R1 reverse-phase column (2.1 mm × 30 mm, 20 µm particle size, 4,000 Å pore size) and desalted. The mobile phase flow rate was 300 μl min⁻¹ and the gradient was as follows: 0–3 min, 0% B; 3–4 min, 0–15% B; 4–16 min, 15–55% B; 16–16.1 min, 55–80% B; 16.1–18 min, 80% B. The column was then re-equilibrated at the initial conditions before the subsequent injection. Buffer A contained 0.1% formic acid in water and buffer B contained 0.1% formic acid in acetonitrile.
The mass spectrometer was controlled by Sciex OS v.1.6.1 using the following settings: ion source gas 1, 30 psi; ion source gas 2, 30 psi; curtain gas, 35; CAD gas, 7; temperature, 300 °C; spray voltage, 5,500 V; declustering potential, 80 V; collision energy, 10 V. Data were acquired from 400–2,000 Da with a 0.5 s accumulation time and 4 time bins summed. The acquired mass spectra for the proteins of interest were deconvoluted using BioPharmaView v.3.0.1 (Sciex) to obtain the molecular mass values. The peak threshold was set to ≥5%, reconstruction processing was set to 20 iterations with a signal-to-noise threshold of ≥ 20 and a resolution of 2,500.