Arginine Specific Digestion

Arginine Specific Protease for Sequence Analysis

Proteases are widely used to generate peptides for analysis of proteins with mass spectrometry. The applications include both proteomics and deep characterization of biopharmaceuticals. Traditional proteases are not always ideal due to missed cleavage and sample preparation induced artefacts and for this reason a toolbox of proteases with different specificities are needed. GingisREX is an arginine specific protease digesting protein C-terminally to arginine residues. The resulting peptides are longer compared to tryptic peptides and may result in increased sequence coverage.

 

GingisREX wotkflow - Arginine Specific

Arginine Specific Digest of Insulin

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).

Peak No. Amino acid sequence Expected monoisotopic mass (Da) Measured monoisotopic mass (Da)
1 GFFYTPKA 929.5 929.5
2 FVNQHLCGSHLVEALYLVCGER 2586.4 2586.5
3 GFFYTPK 858.5 858.4
4 FVNQHLCGSH 1190.6 1190.6
5 LVEALYLVCGER + Na 1434.8 1434.5
6 FVNQHLCGSHLVEALYLVCGERGFFYTPKA 3497.9 3496.8

 

GingisREX RgpB 2016 Poster

 

Data GingisREX - Arginine Specific

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.

Robust Sample Preparation in Urea

The conditions for sample preparation may induce artefacts in the sample and it is therefore desired to avoid high temperatures and basic pH for mass spectrometry analysis. It may also be beneficial to avoid multiple steps and to perform digestion and reduction in a one-pot reaction if possible. To explore the robustness and tolerance of GingisREX to chaotropic agents and detergents, the enzymatic activity was tested in a range of agents and evaluated using a substrate assay. The data showed that GingisREX tolerate urea at 6M, Tween up to 1% but the activity was negatively affected by SDC at 0.1% (Table 1). The enzyme showed activity in pH ranging from 5.0 to 9.0 with an optimum between pH 6.5-8.0. Taken together, GingisREX could be used in a one-pot reaction for simultaneous digestion and denaturation in 6M of urea.

 

 

Chaotropic Agents and Detergents Concentration GingisREX™ activity
Urea 6M 95%
Tween 0.1%
1%
95%
90%
SDC 0.1%
1%
80%
70%
pH 5.0
9.0
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