The molecular weight of the new peak at 5.73 min is 7156.5 Da which is consistent with the molecular weight of ODN4 without the 2-nitrobenzyl group (ODN4UV) (Figure ), confirming that the 2-nitrobenzyl group had been removed from ODN4. We then incubated 10 M of ODN4UV with 0.2 U/mL cathepsin B in 25 mM sodium acetate, 5 mM dithiothreitol (DTT) at pH 5.0 (buffer A) at 37 °C for 1 hour to investigate the enzymatic cleavage of the Val-Ala dipeptide in ODN4UV. After incubation, the oligonucleotide was desalted and analyzed by mass spectrometry. As shown in Figures and, ODN4UV shows excellent stability in buffer A after 1 hour of incubation (blue lines). However, after incubation with cathepsin B for 1 hour, ODN4UV was converted to T10-phosphate and Val-Ala-T12 (Figures and, purple lines).