Advanced Analysis Features
This page will discuss some of the more advanced features in UniDec.
UniDec can match the detected peaks to potential species that might be present using the Oligomer and Mass Tools (Ctrl+T). First, click peak detection in the main window to select peaks. When you have the peaks selected, open Oligomer and Mass Tools. The Oligomer Maker window in the center is the hub for defining which species may be present in the sample. A potential species is defined based on five parameters. First, there is a "Base Offset" which is a uniform mass that gets applied to each potential oligomer of that species. Second, there is the "Monomer Mass", which is the mass of the repeating unit. Third and fourth are the minimum and maximum number of potential oligomers respectively. Finally, the fifth parameter is the name of the species.
From these five parameters, UniDec will construct potential masses by adding the base offset to n units of the monomer mass, where n is any number between the min and max number of potential oligomers. For example, nanodiscs contain two MSP belts and different numbers of lipids. We can define a nanodisc by setting the base offset to the mass of two MSP belts and setting the monomer mass as the number of lipids, subject to a reasonable min and max. If you are looking at a homooligomer, you can leave the base offset at 0 and set the monomer mass as the protein mass. If you are looking at protein-ligand binding, you can set the monomer mass to the ligand and either set the base offset to the protein mass or enter the protein mass on a separate line with a min and a max of 1, which will force it to have exactly one oligomer.
You can define oligomers manually by clicking "Add Oligomer Species" or import it from a text file with five columns (1 for each parameter in the table) and any number of potential species. You can clear the whole list by clicking the button or right click a particular species to delete individual things. You can use the Autocorrelation of the zero-charge mass spectrum to find potential repeating units by clicking the button, or you can automatically calculate protein and RNA masses by right clicking a particular line in the table and selecting "Calculate Mass from Sequence". You can also look for potential species on the Common Mass List and directly import them by right clicking and selecting "Add to Oligomer Builder" (these also can be sorted by mass or by type). These common masses can be helpful in finding PTMs, and you can edit the CSV file of the common masses to include your personal favorites. Definitely send any suggestions for common masses to me.
Finally, after you have defined your potential species, you can start to match them to the peaks you selected. This is done by clicking "Match to Isolated Oligomers" or "Match to Mixed Oligomers". Matching to isolated oligomers will only consider one line at a time and will match to any potential oligomer within each line. Matching to mixed oligomers will take all possible combinations of the lines entered in the oligomer builder, including combinations of species from different lines. The Error Tolerance for Matching is set in a box just above the Oligomer Builder table. The results in the table will show the measured peak mass, the closest match from the predicted masses, and the difference in mass between the two. If the absolute value of the difference is less than the Error Tolerance for Matching, it will print the name of the species in the name column with the number of each species annotated. It will also update the name on the main page to reflect this. After you have defined the oligomers, you can now automatically match species by clicking Analysis > Auto Match Peaks or Ctrl+M without going back into the Oligomer and Mass Tools window.
The charge state of larger ions typically depends on their surface area. For a native protein complex, assuming we have a well-folded and reasonably globular protein, we can calculate the predicted charge state from a given mass based on fits to experimental data (https://doi.org/10.1073/pnas.0910126107). UniDec uses the equation: AvgZ = 0.0467 m^(0.533).
The Native Charge Tools calculates how far the actual charge states deviate from the predicted native charge, which we call the Native Charge Offset. Ions with an offset near 0, typically +/- 3, have charge states that resemble native globular proteins.
Ions with negative offsets have lower charges than you would expect for a native complex. This is potentially because they are CID products that have lost charge due to ejection of highly charged monomers (asymmetric dissociation). In this case, the UniDec Native Charge Tools allows you to put in the mass of the ejected monomer, the number of lost monomers (N), and the width of the charge range you want to extract. You can then reconstruct the starting distribution prior to CID.
Ions with higher offsets are likely unfolded in solution or have more extended conformations and thus acquire more charge during ESI.
In any case, you can extract specific slices of the charge offsets to isolate parts of the Mass vs. Charge space that have similar charge offsets but different charges. This allows you to separate folded from unfolded proteins or different CID products.