Extending a Tandem Mass Spectral Library to Include MS2 Spectra of Fragment Ions Produced In-Source and MSn Spectra.

Tandem mass spectral library searching is finding increased use as an effective means of determining chemical identity in mass spectrometry-based omics studies. We previously reported on constructing a tandem mass spectral library that includes spectra for multiple precursor ions for each analyte. Here we report our method for expanding this library to include MS2 spectra of fragment ions generated during the ionization process (in-source fragment ions) as well as MS3 and MS4 spectra. These can assist the chemical identification process. A simple density-based clustering algorithm was used to cluster all significant precursor ions from MS1 scans for an analyte acquired during an infusion experiment. The MS2 spectra associated with these precursor ions were grouped into the same precursor clusters. Subsequently, a new top-down hierarchical divisive clustering algorithm was developed for clustering the spectra from fragmentation of ions in each precursor cluster, including the MS2 spectra of the original precursors and of the in-source fragments as well as the MSn spectra. This algorithm starts with all the spectra of one precursor in one cluster and then separates them into sub-clusters of similar spectra based on the fragment patterns. Herein, we describe the algorithms and spectral evaluation methods for extending the library. The new library features were demonstrated by searching the high resolution spectra of E. coli extracts against the extended library, allowing identification of compounds and their in-source fragment ions in a manner that was not possible before. Graphical Abstract ᅟ.