When PeakErazor starts, the graph window will by default always show the deviation vs. mass graph. You switch to the Mass defect view either by pressing the button in the center at the top of the graph, or you right-click and select ‘Mass defect view’ from the pop-up graph.

If the mass defect spreads to both sides of the x-axis (as in the example above) you have to do a manual calibration first: press Manual then click at one end of the line of dots (use the low mass area) then pull line to the other end of the dots, following the same direction as the dots. Click once again, and the dots should all be centered around the x-axis. You can now press the Auto button, and all dots will be used for a linear calibration and you can now copy the calibrated peak list back onto the clipboard.

If dots fall outside of the +- 125 ppm limit, they are likely to be non-peptide contaminants (matrix, adduct ions) and can be deleted through the Delete button. Perform another round of automatic calibration after deleting peaks.

You have two controls to adjust how the mass defect works: Slope and Offset.
As the mass defect calibration works on the average of all residues, and Lys and Arg have a higher mass defect than most other residues, this leads to boundary effects where small peptides are overrepresented with regards to K/R and long peptides are underrepresented. The result is that the calibration line is no longer linear, hence a slope compensation.
For most proteins, a slope value of 2.5 will yield the best calibration, however, proteins with an unusual composition may go as high as 4.0 or down to 1.5. As you in general don’t know the composition of your target protein, a default value of 2.5 is recommended. The Offset enables you to add an offset to the final calibration. This feature was mainly included for testing purposes, and it is recommended that you set this value to 0.

Site last updated: April 14, 2024