Modulation of Protein Fragmentation Through Carbamylation of Primary Amines

  • Sylvester M. Greer
  • Dustin D. Holden
  • Ryan Fellers
  • Neil L. Kelleher
  • Jennifer S. Brodbelt
Research Article


We evaluate the impact of carbamylation of the primary amines of the side-chains of Lys and the N-termini on the fragmentation of intact protein ions and the chromatographic properties of a mixture of E. coli ribosomal proteins. The fragmentation patterns of the six unmodified and carbamylated proteins obtained by higher energy collision dissociation (HCD) and ultraviolet photodissociation (UVPD) were compared. Carbamylation significantly reduced the total number of protons retained by the protein owing to the conversion of basic primary amines to non-basic carbamates. Carbamylation caused a significant negative impact on fragmentation of the protein by HCD (i.e., reduced sequence coverage and fewer diagnostic fragment ions) consistent with the mobile proton model, which correlates peptide fragmentation with charge distribution and the opportunity for charge-directed pathways. In addition, fragmentation was enhanced near the N- and C-termini upon HCD of carbamylated proteins. For LCMS/MS analysis of E. coli ribosomal proteins, the retention times increased by 16 min on average upon carbamylation, an outcome attributed to the increased hydrophobicity of the proteins after carbamylation. As noted for both the six model proteins and the ribosomal proteins, carbamylation had relatively little impact on the distribution or types of fragment ions product by UVPD, supporting the proposition that the mechanism of UVPD for intact proteins does not reflect the mobile proton model.

Graphical Abstract


Protein Top down MS/MS Ultraviolet photodissociation Carbamylation 



The authors acknowledge the following funding sources: NSF (Grant CHE1402753) and the Welch Foundation (Grant F-1155). S.M.G. acknowledges a graduate fellowship from the American Chemical Society Division of Analytical Sciences. Funding from the UT System for support of the UT System Proteomics Core Facility Network is gratefully acknowledged. This work was supported in part by the National Institute of General Medical Sciences P41GM108569 for the National Resource for Translational and Developmental Proteomics (NRTDP) based at Northwestern University (to N.L.K.).

Supplementary material

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Copyright information

© American Society for Mass Spectrometry 2017

Authors and Affiliations

  • Sylvester M. Greer
    • 1
  • Dustin D. Holden
    • 1
  • Ryan Fellers
    • 2
  • Neil L. Kelleher
    • 2
    • 3
  • Jennifer S. Brodbelt
    • 1
  1. 1.Department of ChemistryUniversity of Texas at AustinAustinUSA
  2. 2.National Resource for Translational and Developmental ProteomicsNorthwestern UniversityEvanstonUSA
  3. 3.Departments of Chemistry, Molecular Biosciences, and the Feinberg School of MedicineNorthwestern UniversityEvanstonUSA

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