The specificity of proteases for mass spectrometry are crucial to obtain high quality data sets and to avoid complex data interpretation. As a model substrate insulin oxidized β-chain was used to compare the enzymatic specificites of GingisREX and ArgC. Insulin oxidized β-chain contains one arginine and one lysine residue. The digested peptides of both GingisREX and ArgC were analyzed on RP-HPLC and mass spectrometry. GingisREX showed no enzymatic activity on the lysine residue or other extra activities even after prolonged incubation overnight and with a high enzyme to substrate ratio (1:5).

However, ArgC displayed primary activity on arginine residue but enzymatic digestion could also be seen at lysine residues. Additional non-specific activity of ArgC was demonstrated at an enzyme to substrate ratio of 1:20 and overnight incubation. This was not detected with GingisREX at the same conditions (Figure 1). The data confirmed the specific activity at arginine residues of GingisREX and showed unspecific digestion at lysines and other residues using Arg-C (Figure 1, Table 1).

Figure 1. Digestion of oxidized β-chain of insulin with GingisREX and Arg-C was performed O/N at 37°C, enzyme to substrate ratio 1:20 (w/w), 20mM cysteine in buffers at pH 7.4 (GingisREX) and pH 7.6 (Arg-C). The peptides were separated on RP-HPLC. Peak masses are presented in Table 1.