The stability of Lynronne-1, Lynronne-2 and Lynronne-3 in the presence of serum was investigated by monitoring the concentrations of the remaining soluble peptides by reverse phase high-performance liquid chromatography (RP-HPLC), as described previously by Nguyen and colleagues.26 Degradation of Lynronne-1, Lynronne-2 and Lynronne-3 in the presence of trypsin was adapted from a previously described method.46 Briefly, trypsin (5 µl of 0.5 ng/ml) and 37.5 µl trypsin activation buffer (50 mM Tris, 2 mM CaCl2, pH 7.8-8) was added to 7.5 µl of 5 mg/ml Lynronne-1, Lynronne-2 and Lynronne-3 and incubated at 37 °C for different time points (0, 1, 3, and 24 h). An aliquot from each reaction mixture at 0 h was prepared to allow matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric verification of the synthetic peptides prior to incubation. Trypsin action buffer (5 µl) and peptides without trypsin was used as negative control. After the various incubation times, the reaction mixtures were acidified by addition of an equal volume (50 µl) of 10% (v/v) trifluroacetic acid (TFA) to stop further enzyme activity. Lynronne-1, Lynronne-2 and Lynronne-3 degradation products were evaporated to dryness and reconstituted in acetonitrile/water/TFA (40/59.5/0.5%, v/v/v). Samples (1 µl) were carefully placed onto a stainless steel (MALDI) target, covered with 1 µl of matrix (53 mM a-cyano-4-hydroxycinnamic acid in acetonitrile/water/TFA, 70/29.97/0.03%, v/v/v) solution before analysis by mass spectrometry.