Electrospray ionization system

Protein spots differentially expressed in the presence of Cu and/or Mn were extracted from the gels with the aid of a scalpel, cut into segments of approximately 1 mm³, transferred to microtubes containing 1 mL of 5% acetic acid (v/v) and subjected to the following steps: dye removal, protein reduction and alkylation and tryptic digestion using 10 ng mL.¹ trypsin solution. The tryptic digestion of the spots was performed using a specific commercial kit (In-GelDigestZP Kit). The peptide sequences in the extracts obtained by the tryptic digestion process were characterized by liquid chromatography tandem mass spectrometry (LC‒MS/MS). Aliquots of the eluted peptide solutions were analyzed using the nanoAcquity UPLC system coupled to the Xevo Q-TOF G2 mass spectrometer (Waters, Manchester, UK) with electrospray ionization system (Waters, UK), which was equipped with HSS T3 column (Acquity UPLC HSS T3 column 75 mm × 150 mm; 1.8 µm, Waters) and operated in positive ion mode. The data obtained were processed using Protein Lynx Global Server (PLGS) version 3.0 and the UniProt databases were used to identify proteins^{34 (link),39 (link)}.

A UPLC System (Waters, USA) equipped with a photodiode array detector was used. An Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm; Waters, USA) was used for the separation. The injection volume was 5 μL at a concentration of 10 mg/mL CBPP extract. The mobile phase consisted of 0.1 % (v/v) formic acid solution (A) and acetonitrile (B) at a flow rate of 0.4 mL/min. A gradient program was used as follows: 0 min, 2 % B; 13 min, 30 % B; 16 min, 50 % B; 25 min, 80 % B; 28 min, 100 % B; 28–30 min, 100 % B. The column temperature was 30 °C.
Accurate mass measurements were collected using a Q-TOF Premier with an electrospray ionization system (Waters, USA). The electrospray capillary voltage was 3.0 kV and 2.5 kV for the positive and negative modes, respectively. The sample cone voltage was 30 V. The nebulization gas was 600 L/h at 350 °C. The cone gas was 50 L/h, and the source temperature was 110 °C. The Q-TOF Premier acquisition rate was 0.1 s, with a 0.02 s inter-scan delay. The instrument was operated with the first resolving quadrupole in a wide pass mode (50 – 2,500 Da).