Augmenting Middle‐Down Sequence Coverage With Trapped Ion Mobility Spectrometry

Middle-down proteomics has emerged as an attractive alternative to conventional bottom-up workflows because it better preserves sequence information and can annotate concurrent post-translational modifications (PTMs). This is facilitated by a limited proteolysis step, which generates larger polypeptides than those typically found in bottom-up workflows. These larger peptides can host multiple PTMs, are less likely to be lost during chromatographic separation, and thus report on a greater percentage of a protein's sequence than bottom-up. The sequencing of these peptides, however, can result in complex tandem mass spectra, overlapping product ions, decreased signal-to-noise ratios, and less efficient sequencing. The coupling of ion mobility spectrometry and tandem mass spectrometry has demonstrated the ability to deconvolute overlapping product ions, improve signal-to-noise ratios, and increase sequence coverage of protein ions. In this work, we utilize trapped ion mobility spectrometry (TIMS) to deconvolute these complex mixtures of product ions and improve the sequence coverage of middle-down peptides. We demonstrate that this TIMS workflow can be effectively coupled with chromatographic separations. We assess four proteins ranging in mass from 12-kDa cytochrome to a 150-kDa immunoglobulin G and find that while the benefits for small proteins like cytochrome c are minimal, substantial increases in peptide coverage of 43.8%, 24.5%, and 39.9% were measured for myoglobin, carbonic anhydrase, and the NIST antibody, respectively. We can conclude that TIMS is an effective method to improve sequence coverage in middle-down workflows.