Tyrosine nitration in proteins is an important post-translational modification (PTM) linked

Tyrosine nitration in proteins is an important post-translational modification (PTM) linked to various pathological conditions. (FT-ICR) mass spectrometer. CID and IRMPD produced more cleavages in the vicinity of the sites of nitration than ECD. However the Rabbit Polyclonal to VEGFR1 total number of ECD fragments was greater than those from CID or IRMPD, and many ECD fragments contained the site(s) of nitration. We conclude that ECD can be used in the top-down analysis of nitrated proteins, but precise localization of the sites of nitration may require either of the slow-heating methods. The significance of tyrosine nitration in proteins has been recognized in a growing number of publications over the past decade.(1) This post-translational modification (PTM) is one of several occurring during oxidative stress caused by radical species.2,3 It has been linked to such pathological conditions as Alzheimers disease,(4) cardiovascular disease,(5) and atherothrombotic diseases.(6)(or cand z) fragment ions. One of the advantages of ECD over the thermal methods is that it provides a more uniform pattern of cleavages along the backbone, with the only exception being cleavage of the N-terminal to proline(28) and thus leads to greater peptide sequence coverage.29,30 Unlike the thermal methods, disulfide bridges are efficiently cleaved by ECD of peptides,(24) and following capture of a second electron, fragments from the peptide segment inside the disulfide loop can be produced. Furthermore, ECD fragments retain labile post-translational modifications,(31) while CID and IRMPD tend to cleave them. Examples of the efficient use of ECD for localizing PTM sites include phosphorylation,32,33 N- and O-glycosylation,34,35 ubiquitination,(36) sumoylation,(37) and others. Nevertheless, there have been observations that ECD is not universally efficient for all possible peptide modifications. We have recently demonstrated that addition Mollugin manufacture of nitration to tyrosine severely inhibits the production of ECD sequence fragments in peptides.(38) A similar effect was reported by the Beauchamp group for benzyl modifications of cysteine which have an electron affinity (EA) of 1 Mollugin manufacture 1.00 eV.(39) Specifically, 3-nitrobenzylcysteine (EA = 1.00 eV) and 3,5-dinitrobenzylcysteine (EA = 1.65 eV), termed electron predators, inhibit peptide backbone cleavage by ECD and the related electron transfer dissociation (ETD) completely.(39) Apparently 3-nitrotyrosine, structurally similar to nitrobenzylcysteine, was also acting as an electron predator in our ECD experiments. However, we demonstrated that ECD of the triply charged nitrated peptides resulted in some singly charged sequence fragments, which may be the products of secondary electron capture.(38) That result indicated that top-down ECD of intact nitrated proteins may be efficient, as multiple electron capture by multiply charged protein ions usually occurs,24,30 the hypothesis which we Mollugin manufacture put to test in this work. In this study we optimize and compare top-down ECD, CID, and IRMPD of nitrated proteins: myoglobin, cytochrome c, and hen egg white lysozyme (HEWL). Our choice of proteins was due to the different behaviors of their un-nitrated forms under ECD. Previously, we have shown that > 14+ cations of unmodified myoglobin fragment extensively under ECD Mollugin manufacture in our instrument.(40) ECD of unmodified cytochrome c does not produce fragments from the vicinity of Cys14 and Cys17, where the heme group is mounted on the protein.40,41 Local lysozyme provides four disulfide bonds, that have to become cleaved by ECD initial to be able to make backbone fragments from the inside from the molecule, i.e., multiple electron catch is required. Hence nitrated myoglobin represents a functional program where ECD could be affected just by the current presence of the nitrated tyrosine, while indigenous Mollugin manufacture lysozyme and cytochrome c represent systems where ECD performance can be suffering from other adjustments furthermore to nitration. For evaluation, ECD, CID, and IRMPD of alkylated and decreased nitrated.