The Charge Variant Analysis workflow allows the user to correlate cIEF traces with peptide mapping data. The workflow searches for PTMs that commonly modify the pI of proteins and then constructs a theoretical cIEF trace and overlays it onto the experimentally collected trace. This allows the user to visually compare the traces and identify species that are not currently accounted for in the peptide mapping search space. The modifications taken into account include: succinamide, pyroGlu, pyroGln, deamidation, proline amidation, C-term Lys loss, C-term Lys, glycation, and salic acid incorporated glycans. All these modifications are already preset within the Charge Variant Analysis with Reconstruction workflow.
The workflow supports any cIEF file in X,Y format such as .arw files, along with mass spectrometry raw files. For the first step in the analysis both file names need to be renamed. Add peptide to the end of the name for the MS raw file and change the file extension to .pI for the cIEF file as shown below:
Both files can then be dragged and dropped into the Samples table.
Next, add protein sequences and configure the Byonic node with appropriate settings including instrument parameters, digestion, modifications including alkylating agent, etc. and create the project.
The workflow will output both a peptide (.blgc) and chromatogram (.bmap) project. The peptide mapping project can be manually validated. Once completed, open the report and view the "reconstruction export" tab. This tab is formatted for importing into the reconstruction feature. To export, select File->Export->Export pivot table content CSV:
The resulting CSV will have the following output format:
It is important to make sure the modification percent at any given position does not add up to greater that 100 percent or an error will occur when creating the reconstruction. Otherwise, the output format can be taken directly into the reconstruction feature:
Once the CSV has been added the protein targets will need to be configured:
The protein target will need to match the construct used in the cIEF trace, in this case a native antibody was used, so 2 counts of the heavy and light chains are added in the Protein Targets table as shown below:
Next, the Gauss Width must be adjusted to better fit cIEF data. 0.01 is a often a good value to use:
Lastly, Mass Offset can be used to align the theoretical cIEF trace over experimentally collected trace. In this case 0.03 was used:
Once applied the reconstruction will be complete: