When target-decoy false discovery rate estimations are inaccurate and how to spot instances

To address problems with estimating the reliability of proteomic search engine results from mass spectrometry fragmentation data, the use of target-decoy database searching has become the de facto approach for estimating a false discovery rate. Several articles have been written about the effects of different ways of creating the decoy database, effects of the search engine scoring, or effects of search parameters on whether this approach provides an accurate estimate, not all agreeing with each other's conclusions. Hence, there may be some confusion about how effective this approach is and how broadly it can be applied. Although it is generally very effective, in this article I will try to emphasize some of the pitfalls and dangers of using the target-decoy approach and will indicate tell-tale signs that something may be amiss. This information will hopefully help researchers become more astute in their assessment of search results.