Top-Down Mass Analysis of Protein Tyrosine Nitration: Comparison of Electron Capture Dissociation with “Slow-Heating” Tandem Mass Spectrometry Methods

Anal. Chem. 2010, 82, 7283-7292

Top-Down Mass Analysis of Protein Tyrosine
Nitration: Comparison of Electron Capture
Dissociation with “Slow-Heating” Tandem Mass
Spectrometry Methods

Victor A. Mikhailov,^t,* Jesus Iniesta,§ and Helen J. Cooper*^,t

School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K., and Department of
Physical Chemistry, University of Alicante, 03080, Alicante, Spain

Tyrosine nitration in proteins is an important post-
translational modification (PTM) linked to various patho-
logical conditions. When multiple potential sites of nitra-
tion exist, tandem mass spectrometry (MS/MS) methods
provide unique tools to locate the nitro-tyrosine(s) pre-
cisely. Electron capture dissociation (ECD) is a powerful
MS/MS method, different in its mechanisms to the “slow-
heating” threshold fragmentation methods, such as col-
lision-induced dissociation (CID) and infrared multipho-
ton dissociation (IRMPD). Generally, ECD provides more
homogeneous cleavage of the protein backbone and
preserves labile PTMs. However recent studies in our
laboratory demonstrated that ECD of doubly charged ni-
trated peptides is inhibited by the large electron affinity of
the nitro group, while CID efficiency remains unaffected by
nitration. Here, we have investigated the efficiency of ECD
versus CID and IRMPD for top-down MS/MS analysis of
multiply charged intact nitrated protein ions of myoglobin,
lysozyme, and cytochrome c in a commercial Fourier trans-
form ion cyclotron resonance (FT-ICR) mass spectrometer.
CID and IRMPD produced more cleavages in the vicinity
of the sites of nitration than ECD. However the 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

* To whom correspondence should be addressed. Helen J. Cooper, School
of Biosciences, College of Life and Environmental Sciences, University of
Birmingham, Edgbaston, Birmingham, B15 2TT, U.K. Phone: +44 (0)121
4147527. Fax: +44 (0)121 414 5925. E-mail: [email protected].

^t University of Birmingham.

* Present address: School of Chemistry, University of Bristol, Bristol, BS8
1TH, U.K.

§ University of Alicante.

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10.1021∕ac101177r © 2010 American Chemical Society

Published on Web 08/02/2010
species.^2,3 It has been linked to such pathological conditions as
Alzheimer’s disease,⁴ cardiovascular disease,⁵ and atherothrom-
botic diseases.⁶ In vivo, the main nitrating agent for this modifica-
tion is thought to be the peroxynitrite anion (OONO^-), formed
in the reaction of the superoxide anion (O₂^-∙) with nitric oxide
(NO∙). The product of tyrosine nitration is 3-nitrotyrosine with
the NO2 group in the ortho-position to the phenol. Other
nitrating agents can also react with tyrosine and its nitration
is regarded as a marker for general nitrative stress.¹ Impor-
tantly, tyrosine nitration is a selective process occurring only
at specific tyrosine residues.^1,7,8 Therefore, a full description of
the biological processes involving nitrotyrosine requires precise
knowledge of the nitration site(s) in the protein(s).

Mass spectrometry (MS) provides reliable tools for both
identification of nitrated proteins and high-resolution identification
of PTM sites in the protein. A combination of ESI with online
separation of proteins by liquid chromatography (LC-MS) has
been used to identify 3-nitrotyrosine residues in complex protein
mixtures.^8-12 While mass shifts of the peptide fingerprints can
be used to identify the nitration in protein segments, only tandem
MS/MS experiments are able to provide precise localization of
PTMs sites on the protein backbone. The mainstream approach
for locating the sites of nitration is to obtain mass fingerprints of
the peptides from a proteolytic digest of a complex protein mixture
or an individual nitrated protein and further analyze the peptides

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