Mascot v2.3 search engine

The in-gel digestion was carried out as described by Katayama et al.^{61 (link)}. Next, samples were re-suspended with Nano-RPLC buffer A. The online Nano-RPLC was employed on the Eksigent nanoLC-Ultra™ 2D System (AB SCIEX). The samples were loaded on C18 nanoLC trap column (100 µm × 3 cm, C18, 3 µm, 150 Å) and washed by Nano-RPLC Buffer A (0.1% FA, 2% ACN) at 2 μL min⁻¹ for 10 mins. An elution gradient of 5–35% acetonitrile (0.1% formic acid) in 90 min gradient was used on an analytical ChromXP C18 column (75 μm × 15 cm, C18, 3 μm 120 Å) with spray tip. Data acquisition was performed with a Triple TOF 5600 System (AB SCIEX, USA) fitted with a Nanospray III source (AB SCIEX, USA) and a pulled quartz tip as the emitter (New Objectives, USA). Based on combined MS and MS/MS spectra, proteins were successfully identified based on 95% or higher confidence interval of their scores in the MASCOT V2.3 search engine (Matrix Science Ltd., London, UK).

Characterization of the peptides was carried out by Sangon Biotech. Co. Ltd. Briefly, the separation of the peptides was performed on an Eksigent nanoLC Ultra 2D system (AB SCIEX) using a ChromXP C18 column (120 Å, 3 μm, 7.5 mm × 150 mm) at 25°C with a mobile phase composed of a mixture of 0.1% formic acid and 2% acetonitrile in water (A) and 0.1% formic acid and 98% acetonitrile in water (B). The dried sample was dissolved in mobile phase A. The flow rate was 2 ml/min. The following gradient was used: 5%–35% B over 90 min. Mass spectrometry was performed on a Triple TOF 5600 system (AB SCIEX) according to the method of Chen et al. (2013). For information‐dependent acquisition, survey scans were conducted in 250 ms, and there were up to 35 product ion scans that could be collected when they exceeded a threshold of 150 counts/s with a 2⁺–5⁺ charge state. Based on the combination of MS and MS/MS spectra, the proteins could be successfully identified when they showed 95% or a higher confidence interval in the MASCOT V2.3 search engine (Matrix Science Ltd.). The identification of peptides was conducted according to the peptide database (http://www.uwm.edu.pl/biochemia/index.php/en/biopep).