Processing parameters for an antibody drug conjugate (ADC) Workflow in Molecule Profiler software 1.3

For research use only. Not for use in diagnostic procedures.

Answer

The antibody drug conjugate (ADC) workflow in Molecular Profiler software 1.3 helps to identify and further characterize cytotoxic drugs, their breakdown products and the linker connected to the entire antibody sequence.

ADCs are designed for targeted delivery of a cytotoxic molecule to diseased or cancerous tissue. ADCs are composed of three components: the antibody used for selective targeting, the cytotoxic drug, and a linker connecting the two. The following steps describe how to set up the processing parameters for the ADC workflow:

Step1: Select the Processing Parameters tile after launching the Molecule Profiler software.

When using a targeted approach for the ADC workflow, the full amino acid sequences for the drug, its linker and the protein are required.

Step 2: Under the processing parameters window, create a new workflow by selecting ADC from the New menu.

Step 3: Enter the cytotoxic drug or payload and linker as a MOL file using the Open Structure button as shown below.
In the example above, the structure of the cytotoxic drug and its linker are shown, where the site of attachment to the antibody – the OH group – is shown in bold and circled in orange. Users must select the site of attachment using their mouse to encircle the functional group, which will then become bolded. Users must then right click on the bolded chemical group to bring up a pop up menu with additional options. “Mark as site of attachment” is one of the options, and once the user marks this site of attachment it will turn purple as shown in the screenshot (see orange circle).

Step 4: Complete the Antibody Details tab under Compound-Specific Parameters tab. 

• Under Protein Sequence, enter either 1) the light chain or 2) the light chain and heavy chains with S-S bonds specified. The format for entering S-S bonds is a format similar to that used with DataExplorer/PeakView software.
• Indicate the enzyme used for digestion (e.g., trypsin).
• Set the site of conjugation to Lys (K) residues.
• Select the type of conjugation as Loss of water because the coupling of the antibody to the payload and linker results in loss of water. If the antibody is conjugated via S-S bonds, then select Loss of H2.
• Specify the maximum amino acid length of the peptide that is produced via the digest. The current limit in the software is 25.

Once the Digest button is selected, a table with all possible tryptic peptide sequences will appear. Note that you cannot set any missed cleavages. 

Step 5: Save and close the Processing Parameters window.


Step 6. Under Workspaces, set up the batch processing page. Select the MS Sample, the folder of the correct project in which the processing method was created, the processing parameters and name the results file.
Select the green Find Metabolites button. Once the samples are processed, select the results file under Workspaces results.