Abstract
Nitration of tyrosine and other aromatic amino acid residues in proteins occurs in the setting of inflammatory, neurodegenerative, and cardiovascular diseases—importantly, this modification has been implicated in the pathogenesis of diverse diseases and the physiological process of aging. To understand the biological consequences of aromatic nitration in both health and disease, it is critical to molecularly identify the proteins that undergo nitration, specify their cognate modification sites and quantify their extent of nitration. To date, unbiased identification of nitrated proteins has often involved painstaking 2D-gel electrophoresis followed by Western Blotting with an anti-nitrotyrosine antibody for detection. Apart from being relatively slow and laborious, this method suffers from limited coverage, the potential for false-positive identifications, and failure to reveal specific amino acid modification sites. To overcome these shortcomings, we have developed a solid-phase, chemical-capture approach for unbiased and high-throughput discovery of nitrotyrosine and nitrotryptophan sites in proteins. Utilizing this method, we have successfully identified several endogenously nitrated proteins in rat brain and a total of 244 nitrated peptides from 145 proteins following in vitro exposure of rat brain homogenates to the nitrating agent peroxynitrite (1 mM). As expected, Tyr residues constituted the great majority of peroxynitrite-mediated protein nitration sites; however, we were surprised to discover several brain proteins that contain nitrated Trp residues. By incorporating a stable-isotope labeling step, this new Aromatic Nitration Site IDentification (ANSID) method was also adapted for relative quantification of nitration site abundances in proteins. Application of the ANSID method offers great potential to advance our understanding of the role of protein nitration in disease pathogenesis and normal physiology.
Highlights
Aromatic nitration is a posttranslational modification that involves the addition of a nitro (NO2) group to the benzene ring of an aromatic amino acid residue in proteins (Alvarez and Radi, 2003).This modification most commonly occurs during pathological conditions where high levels of reactive nitrogen species (RNS) are formed, primarily peroxynitrite (ONOO−), arising from the near diffusion-limited reaction of nitric oxide (NO) with superoxide (O−2 ) (Pacher et al, 2007)
The most widely recognized protein aromatic nitration event occurs on Tyr residues, yielding proteinaceous 3-nitrotyrosine (3-NT), which often accumulates in the setting of neurodegenerative (Ferrante et al, 1999; Castegna et al, 2003; Sacksteder et al, 2006; Sultana et al, 2006; Reynolds et al, 2007) and cardiovascular diseases (Patel et al, 2000; Rubbo et al, 2000; Turko and Murad, 2002; Harrison et al, 2003; Peluffo and Radi, 2007), as well as during the physiological process of aging
The Aromatic Nitration Site IDentification (ANSID) approach used in the present study followed this general strategy (Figure 1), our protocol was optimized and enhanced by the incorporation of novel strategies and reagents that provide increased specificity, sensitivity and relative quantification of protein nitration sites
Summary
Aromatic nitration is a posttranslational modification that involves the addition of a nitro (NO2) group to the benzene ring of an aromatic amino acid residue in proteins (Alvarez and Radi, 2003).This modification most commonly occurs during pathological conditions where high levels of reactive nitrogen species (RNS) are formed, primarily peroxynitrite (ONOO−), arising from the near diffusion-limited reaction of nitric oxide (NO) with superoxide (O−2 ) (Pacher et al, 2007). The most widely recognized protein aromatic nitration event occurs on Tyr residues, yielding proteinaceous 3-nitrotyrosine (3-NT), which often accumulates in the setting of neurodegenerative (Ferrante et al, 1999; Castegna et al, 2003; Sacksteder et al, 2006; Sultana et al, 2006; Reynolds et al, 2007) and cardiovascular diseases (Patel et al, 2000; Rubbo et al, 2000; Turko and Murad, 2002; Harrison et al, 2003; Peluffo and Radi, 2007), as well as during the physiological process of aging (van der Loo et al, 2000; Beal, 2002; Kanski et al, 2005b) This modification has the dual chemical consequences of both increasing the Tyr residue’s steric bulk and decreasing the pKa of its hydroxyl group (from about 10.1 to 7.2)—potentially resulting in altered interactions with protein binding partners and/or perturbed activity of an enzyme (Abello et al, 2009). Since the detection of nitrated proteins is performed using a 3-NT-specific antibody, this approach is unable to identify proteins that are nitrated on aromatic amino acid residues other than Tyr
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