GLP-1 Construct Deuterium Labeling

For analysis of exchange into the indicated GLP-1 constructs, the peptides were dissolved in 25% acetonitrile/water at 50 μM and kept on ice. Deuterium labeling was initiated with an 18-fold dilution into D2O buffer (10 mM potassium phosphate pD 7.01, 100 mM NaCl) at 21°C. After 10 seconds of labeling, the reaction was quenched with the addition of an equal volume of quenching buffer (150 mM sodium phosphate pH 2.48) at 0°C. Samples were then injected onto an in-house packed POROS 20-R2 trap for peptide trapping and desalting for 3 minutes. A Waters nanoACQUITY LC was used to elute each peptide from the trap with a 15%–70% gradient of acetonitrile over 6 minutes at a flow rate of 100 μL/min. Eluant was directed into a Waters Xevo G2 mass spectrometer operated in TOF-only mode for mass analysis. Data were analyzed as described^{31 (link)}. All mass spectra were processed manually using MagTran. The relative amount of deuterium in the GLP-1 constructs was determined by subtracting the centroid mass of the undeuterated form from the deuterated form, at each condition. Deuterium levels were not corrected for back exchange and thus reported as relative.