2.9. Half-life of peptide 9S1R in serum-

The half-life of peptide 9S1R was determined by spiking the peptide into fetal bovine serum (FBS) for exposure times of 30 s and 5, 30, 60 or 90 min. During the exposure, FBS containing the spike peptides were incubated at 37 °C. The exposure was halted, and the peptide was extracted from FBS using protein precipitation. Protein precipitation was accomplished using 75% ice cold acetonitrile, followed by incubation at − 20 °C for one hour, and centrifugation at 9000 rpm for 10 min at − 4 °C. The supernatant was removed and saved for analysis. Peptide samples were analyzed by High pressure liquid chromatography (HPLC) mass spectrometry (MS) using an ultra-high-resolution Quadrupole Time of Flight (QTOF) instrument (Bruker maxis, Bruker Corporation, Billerica, MA, USA). HPLC mobile phase consisted of 18 MΩ H₂O and HPLC grade formic acid and acetonitrile (> 99% purity, Fisher Scientific, Pittsburgh, PA, USA). The electrospray ionization (ESI) source was operated under the following conditions: positive ion mode; nebulizer pressure: 1.2 Bar; flow rate of drying gas (N2): 8 L/min; drying gas temperature: 200 °C; voltage between HV capillary and HV end-plate offset: 3000 V to – 500 V; mass range was set from 250 to 2900 m/z; and the quadrupole ion energy was 4.0 eV. Low concentration ESI tuning mix (Agilent Technologies, Santa Clara, CA, USA) was used to calibrate the system in the mass range. HPLC separation was achieved using a Dionex UltiMate^® 3000 RSLCnano system (Dionex Corporation, Sunnyvale, CA, USA) equipped with a Waters XTerra C18 column (4.6 × 100 mm, 3.5 μm) (Waters Corporation, Milford, MA, USA). The mobile phase was 0.1% formic acid in water (Buffer A) and acetonitrile (Buffer B) with a flow rate of 0.2 ml/min. A linear gradient method was used to separate the mixture starting at 5% acetonitrile and ending at 60% acetonitrile over 20 mins. The sample injection volume was 5 μl. Data were analyzed using the Compass Data Analysis software package (Bruker Corporation, Billerica, MA, USA).