6550 esi q tof mass spectrometer

Peptides were synthesized on a 0.1 mmol scale by automated flow peptide synthesis. Peptide synthesis was performed on ChemMatrix resin with a 4-(4-Hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB) linker (200 mg, 0.5 mmol/g, 100–200 mesh). The first amino acid (1 mmol, 10 equiv.) was manually coupled to the resin with DIC (0.5 mmol, 78 µL) and DMAP (0.01 mmol, 50 µL of a 0.2M solution in DMF) in 3.17 mL of DMF. The resin suspension was incubated overnight (16–24 h), then was drained and rinsed three times with DMF (5 mL). Subsequent amino acids were added by automated flow peptide synthesis. After the syntheses were complete, peptide cleavage and global deprotection was performed with a solution of trifluoroacetic acid, water, ethane dithiol, and triisopropyl silane (94/2.5/2.5/1). Purification was achieved by preparative RP-HPLC with Agilent Zorbax SB-C18 Prep HT column (21.2 mm × 250 mm, 7 μm) at a flow rate of 15 mL/min using a gradient with water and acetonitrile containing 0.1% TFA. Pure HPLC fractions were pooled and lyophilized. The purified peptides were analyzed as 0.01 mg/mL solutions (50:50 CH₃CN in H₂0 with 1% formic acid) by LC/MS on an Agilent 6550 ESI-Q-TOF mass spectrometer equipped with an Agilent Zorbax 300SB-C3 column (2.1 mm × 150 mm, 5 µM) with a 1–91% gradient of CH₃CN in H₂O with 0.1% formic acid and a flow rate of 0.5 mL/min.

Protein (50 ng) was loaded onto an Agilent Zorbax 5 μm 300SB-C3 column (2.1 × 150 mm) and was eluted with a gradient of 1–91% CH3CN in H2O with 0.1% FA and a flow rate of 0.5 mL/min. The protein was detected on an Agilent 6550 ESI-Q-TOF mass spectrometer.