Electron Capture Dissociation Mass Spectrometry of Tyrosine Nitrated Peptides

In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification.Here, we have investigated the electron capture dissociation (ECD) and collision-induced association (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains

Unusual fragmentation of β-Linked peptides by ExD tandem mass spectrometry

Ion-electron reaction based fragmentation methods (ExD) in tandem mass spectrometry (MS), such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) represent a powerful tool for biological analysis. ExD methods have been used to differentiate the presence of the isoaspartate (isoAsp) from the aspartate (Asp) in peptides and proteins. IsoAsp is a β3-type amino acid that has an additional methylene group in the backbone, forming a Cα–Cβ bond within the polypeptide chain. Cleavage of this bond provides specific fragments that allow differentiation of the isomers. The presence of a Cα–Cβ bond within the backbone is unique to β-amino acids, suggesting a similar application of ExD toward the analysis of peptides containing other β-type amino acids. In the current study, ECD and ETD analysis of several β-amino acid containing peptides was performed. It was found that N–Cβ and Cα–Cβ bond cleavages were rare, providing few c and z type fragments, which was attributed to the instability of the Cβ radical. Instead, the electron capture resulted primarily in the formation of a and y fragments, representing an alternative fragmentation pathway, likely initiated by the electron capture at a backbone amide nitrogen protonation site within the β amino acid residues