Table 4. Products of insulin B chain hydrolysis by NEP.

Retention Time
(min.)	Insulin B Chain fragment	Expected Mass	Observed Mass	Cleavage Site
9.1	1–5	643.31	643.24	H5-L6
18.3	1–10	1188.48	1188.39	H10-L11
22.7	17–24	927.42	927.35	Y16-L17+F24-F25
24.3	1–11	1301.6	1301.47	L11-V12
24.7	15–23	1056.51	1056.42	A14-L15+G23-F24
25.4	1–14	1600.75	1600.64	A14-L15
26.2	24–30	872.44	872.39	G23-F24
29.1	17–30	1634.79	1634.64	Y16-L17
31.9	1–16	1876.89	1876.75	Y16-L17
33.3	15–30	1910.93	1910.8	A14-L15
33.6	12–30	2210.87	2209.93	L11-V12
35.5	11–30	2323.17	2323.06	H10-L11
36.6	1–30	3493.67	3493.52	(insulin B chain)

NEP mediated hydrolysis was carried out as described in Table 2 . The reaction was stopped by adding 10 µL of 5% TFA when approximately half of the substrate had been hydrolyzed (180 min.). The acidified reaction mixture was subjected to HPLC as described in figure 1, individual peaks were collected and identified by mass spectral analysis.