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Proteomics newbie here. I am using proteome discoverer software to analyze the fragmentation of a chemically modified single protein. In my understanding, when we set the Fragment mass tolerance to larger value, we should observe more peptide coverage/modification compared with stricter mass tolerance (for example 1Da vs 0.02 Da).I played with the fragment mass tolerance a bit keeping all other parameters constant. To my surprise, I observed better peptide coverage and newer modifications with lower mass tolerance compared to larger value. How this is possible?

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If your data is high mass accuracy, there isn’t a benefit to using low mass accuracy in your search. You should be hitting nearly all of the true peptide fragment ions with your tight tolerances.

When you use larger mass tolerances, you’re going to match more fragment ions for peptides that are incorrect matches. The result is that incorrect peptide hits score higher while correct peptides don’t increase their scores, so it becomes harder to separate your false peptide hits from your true peptide hits. Then, when you go to filter to a given FDR, this manifests as fewer true peptides passing FDR filtering.

MSFragger (within FragPipe) implements a procedure to optimize your fragment tolerances described briefly here: https://www.nature.com/articles/s41467-020-17921-y .

Full disclosure: I’m an author on the above paper.

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  • $\begingroup$ Thanks for the answer. "Then, when you go to filter to a given FDR, this manifests as fewer true peptides passing FDR filtering" I didn't fully understand this text. Could you elaborate a bit? $\endgroup$
    – Science123
    Commented Apr 22 at 14:49

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