2.6 |. Peptide degradation studies

Peptide stability was determined using a modified version of a degradation assay previously described by Cline and co-workers.^[18,22] Briefly, 30 μM peptide solution was incubated in HeLa lysates diluted to a total protein concentration of 2 mg/mL in an assay buffer (10 mM Tris-HCl, pH 7.6) at 37°C in the dark. Aliquots of the reaction mixture were removed at set intervals, at which point further peptidase activity was quenched by heating the aliquots at 90°C for 5 minutes followed by immediately freezing in liquid nitrogen and then storage at −20°C until analysis by HPLC. The zero-minute time point measurements were made using lysates that were heat killed prior to peptide incubation. HPLC analysis was performed with a Waters 616 pump, Waters 2707 Autosampler, and 996 Photodiode Assay Detector which are controlled by Waters Empower 2 software. The separation was performed on an Agilent Zorbax 300SB-C18 (5 μm, 4.6 × 250 mm) with an Agilent guard column Zorbax 300SB-C18 (5 μm, 4.6 × 12.5 mm). Elution was done with a linear 5%−85% gradient of solvent B (0.1% TFA in acetonitrile) into A (0.1% TFA in water) over 40 minutes at a 1 mL/min flow rate with UV detection at 442 nm. Sample chromatograms for RWRWR and III-67B can be seen in Supporting Information Figure S5. Peak areas were calculated by the Waters Empower 2 software by integration of peaks identified using a peak width of 30.00 and a peak threshold of 50.00. Percent intact peptide remaining was calculated by dividing the area of the parent peptide peak at each time point divided by the area of the parent peptide peak at the zero-minute time point. The identity of the parent peptide peak was confirmed using the parent peptide alone and verified with the t = 0 minute chromatogram. Experiments were performed twice. Representative chromatograms are included in Supporting Information Figure S5.