Mass Spectrometry Analysis of 2-Nitrophenylhydrazine Carboxy Derivatized Peptides

Abstract

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.

Selective cleavage upon ETD of peptides containing disulfide or nitrogen–nitrogen bonds

“…To derivatize peptides at the C-terminal, the EDC coupling reaction (see Supplemental Scheme 1) was performed as follows [38,39]. To a 50 ml volume of 100 mM peptide in water, 5 ml of a 5 mg/ml hydrazine reagent in DMF was added.…”

1-(3-Aminopropyl)-3-butylimidazolium bromide for carboxyl group derivatization: potential applications in high sensitivity peptide identification by mass spectrometry

“…Recently, several neutral derivatization reagents [22][23][24] were exploited and further used for the derivatization of carboxyl groups on peptides. Lu et al applied neutral 1-(2-pyrimidyl)piperazine for the derivatization of carboxyl groups of phosphopeptides; both the ionization efficiency and enrichment specificity of phosphopeptides were clearly improved [22].…”

“…Lu et al applied neutral 1-(2-pyrimidyl)piperazine for the derivatization of carboxyl groups of phosphopeptides; both the ionization efficiency and enrichment specificity of phosphopeptides were clearly improved [22]. 2-Nitrophenylhydrazine was developed by Ball et al to derivatize the carboxl groups of peptides and the detection sensitivity of model peptides was improved by 15-fold [23]. In our previous research, piperazine-based derivatives, 1-(2-pyridyl)piperazine, 1-(2-pyrimidyl)piperazine, 1-(4-pyridyl)piperazine and 1-(1-methyl-4-piperidinyl)piperazine were used for peptide derivatization.…”

Recent Advancement of Chemical Derivatization and Its Applications to High Sensitive Analysis of Peptide in Mass Spectrometry

“…Therefore, reaction solvents should be removed to stabilize the derivatized peptides. Ball et al [35] reported the derivatization of the carboxyl groups of peptides with 2-nitrophenylhydrazine. Peptide hydrophobicity was significantly increased via derivatization, and the detection sensitivity of model peptides could be increased over 15-fold.…”