Mass spectrometry analysis of 2-nitrophenylhydrazine carboxy derivatized peptides

Junmei Zhang, Rowaida Al-Eryani, Haydn L. Ball

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Peptides with two or more basic residues, including those with post-translational modifications (PTMs), such as methylation and phosphorylation, can be highly hydrophilic and, therefore, are often difficult to be retained on a reversed-phase (RP) column. In addition, these highly hydrophilic peptides may carry two or more positive charges, which often fragment poorly upon collisionally activated dissociation (CAD), resulting in few sequence-specific ions. C-terminal rearrangement may also occur during CAD. Furthermore, some PTMs are labile and tend to be lost when subjected to CAD as is the case with phosphorylation on serine or threonine. To overcome the difficulties of separation, detection, and fragmentation of highly hydrophilic peptides, we report here the effect of carboxy group derivatization with 2-nitrophenylhydrazine (this strategy will be called NPHylation for simplicity). NPHylation significantly increases the hydrophobicity of the peptides, eliminates C-terminal rearrangement in all cases, and offers enhanced sensitivity in some cases. In addition, the CAD spectra of the resulting NPHylated peptides carry more sequence-specific ions due to significant reduction of sequence scrambling as observed for peptide EHAGVISVL. Furthermore, the different carboxy derivatives of this peptide undergo sequence scrambling to varying degrees, which clearly demonstrates that the C-terminus has a profound effect on peptide fragmentation. Finally, sequence scrambling is a charge dependent phenomenon, which affects CAD of doubly charged peptides far more than their singly charged counterparts.

Original languageEnglish (US)
Pages (from-to)1958-1967
Number of pages10
JournalJournal of the American Society for Mass Spectrometry
Issue number11
StatePublished - Nov 2011


  • C-terminal elimination
  • Collisionally activated dissociation (CAD)
  • Fragmentation pattern
  • Hydrophilic peptide
  • Peptide carboxy group derivatization by NPHylation
  • Peptide hydrophobicity
  • Sequence scrambling

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy


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