Increasing Protein Sequence Coverage with Internal Fragment Ions for Native Top-Down Mass Spectrometry

Native mass spectrometry (MS) of proteins and protein assemblies reveals size and binding stoichiometry. Native MS and native top-down (TD) MS, i.e., fragmentation of the gas-phase protein, can be effective for deriving structural information for soluble and membrane protein complexes, and much of this information can be correlated to the solution-phase structure. Previous work has shown internal fragments (i.e., fragments containing neither terminus) generated by TD-MS can increase sequence coverage of proteins and proteoforms, yield information on disulfide bonds, and gives information on higher order structure. False positive assignment of internal fragments can be problematic, but high measurement mass accuracy and high resolving power can reduce potential false positive assignment. The value of internal fragments for increasing sequence coverage by native TD-MS will be presented for a number of protein complexes and large proteins, including membrane proteins and monoclonal antibodies.