Cell culture, transient transfection, and sample preparation steps were described earlier [28 (link),34 (link)].
The peptide mixtures were analyzed using two LC-MS/MS systems:

Nanoflow HPLC system (Thermo Dionex Ultimate 3000, ThermoScientific) with Acclaim PepMap100 C18 pre-column, 5 mm × 300 μm; 5 μm particles (Thermo Scientific, #160454) and Acclaim Pep-Map RSLC column 15 cm × 75μm, 2 μm particles (Thermo Scientific, #164534) coupled via CaptiveSpray to the QTOF Impact II mass spectrometer (Bruker). Raw LC-MS/MS data were interpreted with the Bruker Compass DataAnalysis (version 4.3) software. The separation gradient was 48 min from 2% to 50% acetonitrile. Flow rates—300 nL/min.

Nano-HPLC (Agilent Technologies 1200) was coupled to an ion-trap mass spectrometer (Bruker 6300 series) equipped with a nanoelectrospray source via protein HPLC Chip (Agilent Technologies, G4240-62001) with 40 nL trap 75 um × 43 mm 5 um 300SB-C18-ZX and analytical column packed with ZORBAX 300SB-C18, 5 µm particle size. The separation gradient was 7 min from 5% to 90% acetonitrile. Flow rates—300 nL/min.

The LC-MS/MS instruments were set to monitor transitions of biotinylated (m/z 648.8, collision energy 33.0 eV) and propionylated (m/z 563.2, collision energy 27.0 eV) forms of BAP peptide in samples.
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