It hit me today: What if the theoretical mass I’m calculating for ions is consistently off? Even if it is just a little (talking fractions of a proton), that would negatively impact a peptide matching algorithm’s performance.

How can we measure this?

Relatively easy. The somewhat challenging part is getting a set of spectra from your machine where you know the peptide from which each spectrum came. These tests sets are very useful and finding consistent theoretical peak error is one of them.

To calculate average theoretical peak error, go through each spectrum you have. For each spectrum, take the peptide it was derived from and find the theoretical ions of this peptide. Then, for each of these theoretical ions, find the most intense (it’s so intense!) peak of the spectrum that falls within a fairly conservative mass window. (This mass window should be large enough to account for normal error but small enough to avoid false matches. A good starting point is 0.5Da.) Find the mass difference between this peak and the theoretical ion.

Do this for every theoretical ion of every peptide for every spectrum and get the average.

Voila!