Xianquan Zhan 1, Ying Long1 and Dominic M. Desiderio2
- 1 Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha, Hunan, P R. China
- 2 The Charles B. Stout Neuroscience Mass Spectrometry Laboratory, Department of Neurology, College of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, USA
Overview of Tyrosine Nitration
Tyrosine nitration of a protein is an addition of a nitro group (-NO2) to position-3 of the phenolic ring of a tyrosine residue in a protein. Tyrosine nitration is a relatively chemically stable oxidative/nitrative modification and is a marker of oxidative injuries. Tyrosine nitration alters the functions of proteins associated with multiple physiological and pathological processes such as cancer, inflammation disease, and neurodegenerative diseases [1-7]. Tyrosine nitration occurs in a normal physiological status and increases in a pathology [3, 8]. Tyrosine nitration changes physical and chemical properties relative to a tyrosine [2, 9, 10]. A nitro group is an electron-withdrawing group that decreases the electron density of the phenolic ring of a nitrotyrosine relative to a tyrosine. The decreased electron density decreases the affinity between enzyme- substrate, ligand-receptor, or antigen-antibody when tyrosine nitration occurs within these binding regions . Tyrosine nitration also alters the pKa of the phenolic hydroxyl group of a nitrotyrosine (pKa = ~7.1) significantly lower than that of tyrosine (pKa = ~10) [2, 9], and the spectrophotometric properties of a nitrotyrosine residue are different from tyrosine. A nitrotyrosine can be reduced to a stable aminotyrosine , which is useful for further study (discussed later). Tyrosine nitration is known to occur within a tyrosine kinase phosphorylation motif ([R/K]-XX-[D/E]-XXX-Y or [R/K]-XXX)-[D/E]-XX-Y; Y = the phosphorylation site) to impact on the tyrosine phosphorylation signaling system that is important in pathologies [11-16]. Moreover, an in vivo denitrase indicates that tyrosine nitration and denitration are a reversible
Analysis of Protein Post-Translational Modifications by Mass Spectrometry, First Edition. Edited by John R. Griffiths and Richard D. Unwin.
© 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.
dynamic reaction similar to phosphorylation and dephosphorylation [17, 18]. Therefore, tyrosine nitration not only results from oxidative injuries but also alters protein structure and function. It involves multiple biological consequences, for example, sensitivity to proteolytic degradation, modification of enzymatic activities, impact on protein tyrosine phosphorylation, immuno- genicity, and implication in disease [19-23].
Characterization of tyrosine nitration and accurate determination of each nitration site are essential to address biological functions and roles of tyrosine nitration . However, it is very challenging to identify tyrosine nitration due to the varied mass spectrometry (MS) behaviors of a nitro group, its extreme low abundance in vivo [24, 25], and limited MS sensitivity [2, 26-29]. Selection of an appropriate MS, in combination with chemical derivation  and preferential enrichment [2, 3, 30], is needed to identify tyrosine nitration . The varied MS behaviors of a nitro group involve a characteristic photodecomposition pattern of a nitro group in UV-laser-based matrix-assisted laser desorp- tion/ionization (MALDI)-MS analysis of a nitroprotein [27-29] but not in electrospray ionization (ESI)-MS [27, 31-35]. This photodecomposition pattern of a nitro group decreases signal intensities of a nitropeptide and complicates the interpretation of a MALDI-MS spectrum. However, the characteristic photodecomposition pattern can confirm the existence of a nitro group with MALDI-MS . A nitrotyrosine residue is easily reduced to a more stable aminotyrosine residue for MS analysis . Thus, chemical derivation is helpful for MS analysis. The in vivo low abundance of endogenous nitrotyrosine sites (1 in 106 tyrosine residues) and the MS sensitivity requirement require a preferential enrichment of nitroproteins or nitropeptides from a biological extract before MS analysis [26, 36, 37].
Several chemical derivation and targeted enrichment approaches have been published : (i) antinitrotyrosine antibody-based immunoaffinity enrichment of nitropeptides  or nitroproteins [2, 40]; (ii) use of selective chemo- precipitation and subsequent release of tagged species (conversion of nitro group to a small 4-formylbenzylamido tag) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of nitropeptides ; (iii) conversion of a nitro group to an amino group coupled with targeted enrichment  by first acetylating free amines, followed by conversion of nitrotyrosine to amino- tyrosine, and then biotinylation of aminotyrosine; (iv) conversion of a nitro group to an amino group coupled with derivation of the amino group . Briefly, protection of a- and e-amino groups in a protein or peptide with 13C0/13C4- or D0/D6-acetic anhydride, reduction of nitrotyrosine to aminoty- rosine with sodium dithionate (also known as sodium hydrosulfite), and derivation of aminotyrosine with 1-(6-methyl[D0/D3]nicotinoyloxy) succinim- ide; (v) reduction of the nitro group to an amino group and dansylate with dansyl chloride, followed by MSn analysis [44, 45]; (vi) a new quantitative identification strategy used isobaric tags for relative and absolute quantification
(iTRAQ) reagents to selectively label nitrotyrosine residues (not primary amines) coupled with MS analysis ; (vii) after the use of “light”- and “heavy”-labeled acetyl groups to block N-terminal and lysine residues of tryptic nitropeptides, reduction of nitrotyrosine to aminotyrosine with sodium dithionite and derivatization of light- and heavy-labeled aminotyrosine peptides with either tandem mass tags (TMT) or iTRAQ, respectively ; and (viii) combining fractional diagonal chromatography (COFRADIC) [48,49]- peptide sorting, which is based on a hydrophilic shift after the reduction of the nitro group to its amino counterpart, with ESI-MS  and MALDI-MS . Except for the proteomics method based on antinitrotyrosine antibodies and gel-based separation, chemical derivation, precursor ion scanning, and multidimensional chromatography have been used to characterize and quantify tyrosine nitration in a protein and in its modification sites [37, 50].
In-depth analysis of tyrosine nitration in a protein is necessary to address fully the biological functions and roles of tyrosine nitration, and several aspects should be considered here . It is important to be able to quantify tyrosine nitration in a protein in a pathological status and the degree of nitration with quantitative proteomics . Quantification of body fluid biomarkers (nitro- protein, nitropeptide) is important for prediction, diagnosis, and prognosis of a disease with quantitative body fluid nitroproteomics and nitropeptidomics [26, 51]. It is also important to be able to locate nitrotyrosine sites within an important protein domain and motif with bioinformatics [2, 52], to clarify important protein system networks that involve nitroproteins with systems biology [26, 53], and to reveal the three-dimensional structure of a nitroprotein to address the influences of local primary structure on tyrosine nitration . Finally, it is important to also discover the effects of tyrosine nitration on protein function toward development of a drug against tyrosine nitration [26, 55].