Acquity uplc

The detailed synthesis of each analog is described in Supplementary Materials. Deuterated chloroform or dimethyl sulfoxide was used as a solvent. For purity analysis, an Agilent Acquity UPLC or HPLC were used. All compounds were dissolved in methanol for purity analysis (Supplementary Figure S2). ¹H NMR and ¹³C NMR spectra were recorded on an Agilent 400 MHz NMR spectrometer (Supplementary Figure S3).

Initial analysis of Fc-peptide products was carried out by reversed-phase HPLC/MS of the intact or reduced (5 mM DTT, 55 °C, 30 min) product on the system described below. However, mass accuracy and measurement precision were insufficient to ensure unambiguous determination of the state of the peptide carboxy terminus. Consequently, samples were digested with HRV3C protease (PreScission, GE Healthcare Bio-Sciences, Pittsburgh, PA). Digestions of Fc-peptide were performed for 5 h at 5 °C in a buffer consisting of 20 mM TrisHCl, pH 7.0, 150 mM NaCl, 1 mM DTT using one unit of enzyme for up to 100 μg of protein. Polypeptides resulting from the digestion were chromatographically resolved on a Waters (Milford, MA) Acquity UPLC with an Agilent (Santa Clara, CA) PLRP-S column (2.1 × 50 mm, 5 μ d_p, 1000 Å) using a multi-linear gradient. Buffer A was 0.1 % formic acid, and buffer B was 0.1 % formic acid in acetonitrile. The peptides were mass analyzed on a Waters API US mass spectrometer scanned from m/z 500–3000 at a rate of 1 Hz. The peptide m/z data was deconvoluted to the single-charge mass domain by the MaxEnt3 algorithm implemented in the Waters MassLynx data system.

UPLC-QqQ/MS analysis was carried out on a triple-quadrupole tandem mass spectrometer coupled to an Agilent Acquity UPLC (Agilent, Santa Clara, CA, USA). Chromatographic separations were performed on an Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm). The gradient elution program adopted two elution solvents, A (water: formic acid; 99.9:0.1, v/v) and B (acetonitrile): 0 min (A 95%:B 5%), 15 min (A 75%:B 25%) and 18 min (A 0%:B 100%). The other major settings were as follows: the flow rate of the mobile phase, 0.3 mL/min; detect wavelength, 280 nm; temperature, 30 °C; the injection volume, 0.5 μL.
The detection of MLPE was operated in the multiple reaction monitoring (MRM) mode using electrospray negative ionization (ESI⁻). The QqQ/MS parameters were set as follows: source temperature 300 °C; nebulizer pressure 45 psi; gas flow 5 L/min; capillary voltage 3.5 KV (ESI⁻); gas temperature 350 °C; cell accelerator voltage 5 V.