Improved Annotation of Internal Fragments via Trapped‐Ion Mobility Enhanced Top‐Down Sequencing of Protein Ions

Analysis of intact protein ions allows for valuable insights into complex biological processes. However, top-down mass spectrometry data is often convoluted and frequently results in numerous overlapping product ions. Ion mobility spectrometry (IMS) can aid in the deconvolution of these spectra, and we previously demonstrated that the IMS separations provided by trapped IMS (TIMS) significantly increased the sequence coverage provided by collision induced dissociation (CID). In this work, we further improve the "CIDtims" method by incorporating the dynamic control of ion populations, optimizing the bioinformatic approach to better leverage the mobility separation, and finally porting the technique to an improved version of the instrument, the timsTOF Pro2. Lastly, we utilize these improvements to assess internal ions generated by CIDtims. Internal ions are of particular importance in the CIDtims workflow as all charge states are simultaneously activated and the highest charge states will likely be subjected to "over fragmentation." We demonstrate that mobility separation increases the signal-to-noise ratios and the isotopic fit scores of internal ions and enables the assignment of additional product ions.