Analyzing internal fragmentation of electrosprayed ubiquitin ions during beam-type collisional dissociation

Kenneth R. Durbin, Owen S. Skinner, Ryan T. Fellers, Neil L. Kelleher*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

29 Scopus citations


Gaseous fragmentation of intact proteins is multifaceted and can be unpredictable by current theories in the field. Contributing to the complexity is the multitude of precursor ion states and fragmentation channels. Terminal fragment ions can be re-fragmented, yielding product ions containing neither terminus, termed internal fragment ions. In an effort to better understand and capitalize upon this fragmentation process, we collisionally dissociated the high (13+), middle (10+), and low (7+) charge states of electrosprayed ubiquitin ions. Both terminal and internal fragmentation processes were quantified through step-wise increases of voltage potential in the collision cell. An isotope fitting algorithm matched observed product ions to theoretical terminal and internal fragment ions. At optimal energies for internal fragmentation of the 10+, nearly 200 internal fragments were observed; on average each of the 76 residues in ubiquitin was covered by 24.1 internal fragments. A pertinent finding was that formation of internal ions occurs at similar energy thresholds as terminal b- and y-ion types in beam-type activation. This large amount of internal fragmentation is frequently overlooked during top-down mass spectrometry. As such, we present several new approaches to visualize internal fragments through modified graphical fragment maps. With the presented advances of internal fragment ion accounting and visualization, the total percentage of matched fragment ions increased from approximately 40% to over 75% in a typical beam-type MS/MS spectrum. These sequence coverage improvements offer greater characterization potential for whole proteins with no needed experimental changes and could be of large benefit for future high-throughput intact protein analysis.

Original languageEnglish (US)
Article number1078
Pages (from-to)782-787
Number of pages6
JournalJournal of the American Society for Mass Spectrometry
Issue number5
StatePublished - Feb 26 2015


  • Internal fragmentation
  • Proteomics
  • Top-down mass spectrometry

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy


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