Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins

  • David J. Foreman
  • Eric T. Dziekonski
  • Scott A. McLuckeyEmail author
Focus: Honoring Carol V. Robinson's Election to the National Academy of Sciences: Research Article


A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500–2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa.

Graphical Abstract


Ion/ion reactions Intact protein identification Top-down Selective fragmentation 



D.J.F. would like to acknowledge Catherine Rawlins and Daniel Donnelly of the Agar Lab at Northeastern University for helpful discussion regarding top-down sample preparation.

Funding Information

This work was supported by the National Institutes of Health (NIH) under Grant GM R37-45372. Graduate student support for D.J.F. provided by W. Brooks Fortune Fellowship in Analytical Chemistry.

Supplementary material

13361_2018_1965_MOESM1_ESM.docx (341 kb)
ESM 1 (DOCX 340 kb)


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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Department of ChemistryPurdue UniversityWest LafayetteUSA
  2. 2.SCIEXConcordCanada

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