Evaluation of the performance of PepQuery for validating completely novel protein sequences. (A) Sensitivity and (B) specificity evaluation for novel peptide sequence validation. Mod. filtering means unrestricted modification searching-based filtering. (C) A spectrum matched to a gold-standard negative peptide without unrestricted modification searching-based filtering. Blue and red colors indicate matched peaks, whereas gray indicates unmatched peaks. (D) The same spectrum in C can be matched to a reference peptide with an ammonium salt modification on aspartic acid with very few unmatched peaks and a high score.