IdeS Protease is an immunoglobulin-degrading enzyme from Streptococcus pyogenes (IdeS). It cleaves Immunoglobulin G (IgG) with high specificity at a single site below the hinge region (G|G), yielding F(ab’)2 and Fc fragments.
After IdeS digestion, a reduction step can be performed for analysis of LC, Fd, and Fc/2 subunits. Byos Intact Mass can analyze IdeS digested samples by inputting the LC, Fd, and Fc/2 sequences in the Chains table or by inputting the heavy and light chain sequences and using the Clipping feature.
Input Fc/2, Fd, and LC sequences:
Under the Sequences and masses tab, import a fasta file containing separate sequences for Fc/2, Fd, and LC to the Chains table. This will automatically populate the Sequence combinations table.
The Fc/2, Fd, and LC sequences can also be added row by row to the Chains table by clicking "Add" and copying the sequence for each subunit into separate rows. Check the Mirror Chains table box to copy the Fc/2, Fd, and LC subunits from the Chains table to the Sequence combinations table. The Mirror Chains table must then be unchecked to edit Sequence combinations further.
If disulfides are fully reduced, set the Disulfides column in the Sequence combinations table to "Disulfide count = 0" for all three sequences.
Example Sequences and masses table for reduced IdeS analysis:
Input heavy and light chain sequences with the Clipping feature:
Add the heavy chain and light chain sequences to Chains table and Sequence combinations table as described in section above. If disulfides are fully reduced, set the Disulfides column in the Sequence combinations table to "Disulfide count = 0" for both sequences.
Example Sequences and masses table for reduced IdeS analysis with Clipping feature:
IdeZ and IdeS have the same specificity; both cleave at the exact same single site below the hinge region of IgG. However, IdeZ is more versatile as it cleaves mouse IgG2a more efficiently than IdeS.
human lgG1, lgG3, lgG4: CPAPELLG|GPSVF
human lgG2: CPAPPVA|GPSVF
murine lgG2a: CPAPNLLG|GPSVF
murine lgG3: CPPGNILG|GPSVF
IdeZ Protease efficiently cleaves human, humanized, chimeric, sheet, rabbit and monkey IgG as
well as mouse IgG2a and IgG3. IdeZ Protease will also cleave Fc-fusion proteins, such as Enbrel.
IdeZ Protease does not cleave mouse IgG1 or IgG2b, rat, porcine, bovine or goat IgG. It also does not
cleave non-IgG isotypes including IgA, IgM, IgD and IgE.