A spectroscopic test suggests that fragment ion structure annotations in MS/MS libraries are frequently incorrect.

Modern untargeted mass spectrometry (MS) analyses quickly detect and resolve thousands of molecular compounds. Although features are readily annotated with a molecular formula in high-resolution small-molecule MS applications, the large majority of them remains unidentified in terms of their full molecular structure. Collision-induced dissociation tandem mass spectrometry (CID-MS²) provides a diagnostic molecular fingerprint to resolve the molecular structure through a library search. However, for de novo identifications, one must often rely on in silico generated MS² spectra as reference. The ability of different in silico algorithms to correctly predict MS² spectra and thus to retrieve correct molecular structures is a topic of lively debate, for instance in the CASMI contest. Underlying the predicted MS² spectra are the in silico generated product ion structures, which are normally not used in de novo identification, but which can serve to critically assess the fragmentation algorithms. Here we evaluate in silico generated MSⁿ product ion structures by comparison with structures established experimentally by infrared ion spectroscopy (IRIS). For a set of three dozen product ion structures from five precursor molecules, we find that virtually all fragment ion structure annotations in three major in silico MS² libraries (HMDB, METLIN, mzCloud) are incorrect and caution the reader against their use for structure annotation of MS/MS ions. Collision-induced dissociation tandem mass spectrometry offers to resolve molecular structures through library searches, however, for de novo identifications, one must often rely on in silico-generated spectra as reference. Here, the authors evaluate in silico generated MSⁿ product ion structures by comparison with spectroscopically established structures and find that for 36 randomly selected MS/MS product ions, the vast majority of annotations in three major libraries are incorrect, primarily due to unaccounted for cyclization rearrangements. [ABSTRACT FROM AUTHOR]