Recent studies have suggested that adenosine 5′-monophosphate (AMP) post-translational modification of proteins could represent a novel molecular signaling pathway. Mass spectrometric fragmentation characteristics of this modification have not previously been described and studied systematically. In this work, we therefore examined the fragmentation pattern of chemically synthesized peptides containing AMPylated Thr, Ser, and Tyr. The formation of characteristic ions and the influence of collision energy (CE) on the detection of characteristic ions and their relative peak intensity are reported. When peptide with AMPylated Ser/Thr underwent collision induced dissociation (CID), peaks at m/z 348.1, 136.1, and 250.1, fragments with AMP group attached, and fragments consistent with neutral loss of 347 Da were major characteristic ions; fragments consistent with neutral loss of 135 Da or 249 Da were weaker and not always detectable. The observations for Tyr AMPylation followed the same general patterns as those for Ser/Thr modification, with the exception that the ions detected for Tyr AMPylation did not include either the peak at m/z 348.1, or fragments with a mass shift of −347 Da. The results described in this paper highlight a series of diagnostic ions, which can be used not only to confidently identify the AMPylation site based on MS and MS/MS data, but also to selectively scan AMPylated peptides in complex protein mixtures.

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.