The reaction was started by adding one microlitre of enzyme
solution of an appropriate concentration (to achieve the desired
enzyme concentration) to ten microlitres of substrate solution. Samples of
0.5 
l were taken and mixed with 1.0 l
matrix solution (see below) on the sample plate. The addition of
3-hydroxypicolinic acid stops the enzymatic
reaction[42].
Spectra were acquired in reflectron mode using the parameters given in table 2.2. Insulin was used as external standard, calibration was performed using the expected signals of substrate hydrolysis as internal standard. The sum of at least 16 laser shots were used.
| Acceleration voltage | 20,000V |
| Delay Time | 300ns |
| Grid Voltage | 58% |
| Guide Wire Voltage | 0.150% |