REVERSIBLE POST-TRANSLATIONAL MODIFICATION OF PROTEINS BY NITRATED FATTY ACIDS IN VIVO

Nitric oxide ((*)NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon beta to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9- and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC(50) of approximately 3 microM for both nitroalkenes, an IC(50) equivalent to the potent thiol oxidant peroxynitrite (ONOO(-)) and an IC(50) 30-fold less than H(2)O(2), indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post-translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling, and protein trafficking.


cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible posttranslational modification of nucleophilic amino acids in GSH and GAPDH in vivo.
The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these macromolecules, both inducing translocation to membranes and explaining why these abundant derivatives had not been previously detected via traditional HPLC analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post-translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling and protein trafficking.
Nitric oxide (⋅NO) exerts a broad influence on cell and inflammatory signaling via both cGMP-dependent and -independent oxidative, nitrosative and nitrative reactions (1,2). The nitration of polyunsaturated fatty acids present in both membranes and lipoproteins is now emerging as a novel mechanism for transducing ⋅NO-dependent redox signaling (3,4). Recent evidence indicates that all major unsaturated fatty acids present in human blood contain some proportion of alkenyl nitro derivatives, [R 1 HC=C(NO 2 )R 2 ], also termed nitroalkenes. Due to the prevalence of fatty acid nitroalkenes in healthy humans, these species are now appreciated as the single largest pool of bioactive oxides of nitrogen in the vasculature (5). The two most clinically abundant nitroalkene fatty acid derivatives, nitro-oleic acid (9-and 10-nitro-9-cisoctadecaenoic acids; OA-NO 2 ) and nitro-linoleic acid (10-nitro-9,12-octadecadienoic and 12-nitro-9,12-octadecadienoic acids; LNO 2 ) are present in net concentrations of >1 μM in membrane and lipoprotein lipid extracts prepared from healthy human blood. These nitroalkene concentrations far exceed the <10 nM concentrations reported for other NO derivatives, including S-nitrosothiols, nitrosyl heme and 3-nitrotyrosine, and the <300 nM concentrations reported for nitrite (NO 2 -, (6)).
Fourth, thrombin-induced platelet aggregation is inhibited by nitroalkene-induced attenuation of cAMPdependent Ca +2 mobilization and activation of the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at Ser-157 (8). Current evidence supports a dual regulation of platelet adenylyl cyclase and phosphodiesterase E activities by nitroalkenes. Finally, fatty acid nitroalkene derivatives, as opposed to native unsaturated fatty acids, potently regulate the expression of key inflammatory, cell proliferation and cell differentiation-related proteins (5,9).
The broad impact of nitroalkenes on differentiated characteristics of cells and tissues motivated analysis of additional chemical reactivities of nitroalkenes that would account for their pluripotent signaling capabilities. NMR, MS/MS and spectroscopic analysis of nitrated fatty acids indicates the presence of predominantly nitroalkene rather than nitroalkane derivatives in clinical specimens, with this structural configuration conferring unique biochemical and pharmacological qualities.
The alkenyl nitro configuration of endogenous nitrated fatty acids indicates potential electrophilic reactivity of the βcarbon adjacent to the nitro-bonded carbon. This would promote nitroalkene reactivity with nucleophiles (i.e. Cys and His residues) via Michael addition reactions, yielding new carboncarbon or carbon-heteroatom bond frameworks (2,10). This electrophilic property of nitroalkenes was first suggested by the biological detection of α,β-nitro-hydroxy fatty acid derivatives (5). Also, synthetic nitroalkenes generated hydroxy derivatives under aqueous conditions, presumably induced by reaction with the low levels of hydroxide ion present at physiological pH (2).
The electrophilic HNE reacts with sulfhydryl groups (12), the imidazole of histidine (13) and the ε-amino of lysine (14). Covalent modification by electrophilic lipids has been shown to alter the structure and activities of cathepsin B (15), Keap1 (16) and insulin (13).
Herein, the reactions of nitroalkenes with cysteine and glutathione (GSH) are characterized, and GAPDH is evaluated as an exemplary target protein for nitroalkene reaction. GAPDH is of additional relevance, as a) catalytic thiols are essential for GAPDH activity (17), b) GAPDH can be inhibited by electrophilic lipids (18,19) and c) the ⋅NO-dependent post-translational modification and translocation of GAPDH to specific cellular microenvironments impacts on the roles of GAPDH in intermediary metabolism and apoptotic signaling (20,21).
We reveal herein, via model reaction systems and the proteomic analysis of human red cells, that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and proteins. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that fatty acid nitroalkene derivatives serve to regulate protein function and trafficking.
GSH -nitroalkene reactions. GSH (1 mM) solvated in 50 mM sodium phosphate buffer (pH 7.4) was treated with equimolar LNO 2 or OA-NO 2 at 20°C for 30 min. The reaction mixture was analyzed by ESI-MS with and without liquid chromatography. When the adduct was analyzed by reverse phase HPLC-MS, the elution protocol used for tryptic peptide resolution was employed (see below). Synthetic nitroalkene adducts of GSH were used as standards for LC separations and quantitative MS analysis, including GS-OA-NO 2 , GS-[ 13 C 18 ]OA-NO 2 , GS-LNO 2 , GS-[ 13 C 18 ]LNO 2 . These derivatives were prepared by reacting the corresponding nitroalkene (2 mM) with GSH (20 mM) in sodium borate buffer (0.1 M), pH 10.3 for 30 min at 20°C. GS-nitroalkene adducts were purified from non reacted GSH by reverse phase chromatography. Samples were loaded onto PrepSep™ C18 columns (Fisher Scientific, Pittsburgh, PA) equilibrated with 0.1% formic acid. After washing, adducts were eluted with 0.1% formic acid in methanol and fractions concentrated in vacuo. The concentration of purified standards was determined by elemental nitrogen analysis following pyrrolysis, using an Antek chemiluminescent nitrogen detector (Houston, TX).
Matrix-assisted laser desorption and ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. MALDI-TOF MS was performed using a Voyager DE PRO system (Applied Biosystems, Foster City, CA), equipped with a N 2 laser source (337 nm).
Mass spectra were acquired for positive ions in linear and reflector mode. For whole molecular mass determinations, native and nitroalkene-treated GAPDH (0.5 μM) was desalted by using C18-ZipTip (P10) (Millipore Corporation, Bedford, MA), following manufacturer protocols. Proteins were eluted by adding the matrix solution (sinapinic acid, 10 mg/ml in 50% acetonitrile (ACN), 0.2% TFA) and directly applied to the stainless steel sample plate. GAPDH-derived peptide masses were measured in the reflector mode with an accuracy of ~50 ppm, attained by internal mass calibration using characteristic GAPDH tryptic peptides as mass standards and α-cyano-4-hydroxycinnamic acid as the matrix. To obtain complete post-source decay (PSD) spectra, a series of reflectron mass spectral segments were acquired, each optimized to focus fragment ions within different mass/charge (m/z) ranges. Each segment was stitched together using Biospectrometry Workstation software to generate a composite PSD spectrum (26). Peptide mapping and electrospray ionization liquid chromatography-mass spectrometry analysis (ESI-LC-MS). Native and nitroalkenemodified GAPDH (10 μM) were digested with sequencing grade trypsin in 50 mM pyrophosphate buffer, pH 7.4 at 37°C for 20 h using an enzyme:substrate ratio of 1:50 (w/w). Peptide samples were analyzed by ESI-LC-MS using a LCQ ion trap mass spectrometer (LCQ Deca, Thermo Finnigan, San Jose, Ca). A reversedphase column (5 μm, 2.1 x 150 mm, 300 Å, from Grace Vydac, (Hesperia, CA) was eluted with Solvent A (0.1% formic acid) and solvent B (0.08% formic acid in acetonitrile). Peptides were eluted at 40°C with a linear gradient of solvent B (2 to 60 % in 105 min) at a flow rate of 0.25 ml⋅min -1 . Electrospray voltage was 5 kV and capillary temperature was 260°C. Peptides were detected in the positive ion mode using a mass range of 100 -2000, following mass calibration with the GS-OA-NO 2 adduct. MS/MS peptide analyses was perfomed by nanospray ion trap mass spectrometry (LTQ, Thermo Electron, San Jose, CA) using a BioBasic C18 picofrit column, 5µm, 75µm x 10cm (Thermo Electron), and a Zorbax 300SB-C18 trap column, 5µm, 0.3 x 50mm (Agilent Technologies, Chicago) with a flow rate of 700nL⋅min -1 and similar gradient conditions.

GAPDH -liposome interactions.
A lipid film was prepared by dissolving soybean phosphatidylcholine (200 mg) and cholesterol (50 mg) in chloroform and removal of solvent under a stream of nitrogen. Multilamellar vesicles were prepared by hydrating the lipid film with 10 ml of 10 mM pyrosphophate buffer, pH 7.4 at 25°C for 1 hr. After vortexing, the suspension was place in a 25°C sonicating water bath for 30 min. Subsequently, control or nitroalkene treated GAPDH preparations (100 μl of 10 mg/ml protein) were incubated with liposome suspensions (1 ml) for 30 min at 25°C. Liposome-associated GAPDH was sedimented by ultracentrifugation at 100,000xg for 30 min. Soluble GAPDH in the supernatant and sedimented liposome-associated GAPDH was analyzed by SDS-PAGE electrophoresis (4-12% BI-Tris, Criterion TM Precast Gel, Bio-Rad, Hercules, CA), stained with Coomasie Blue and quantitated by densitometry using AlphaInotech software.
Identification of endogenous nitroalkylated GSH in human red blood cells. All human studies were reviewed and approved by the UAB Institutional Review Board (Protocol #X040311001). Human red blood cells (RBCs) were obtained by centrifugation (800xg for 10 min) of freshly drawn heparinized blood from a healthy donor. RBCs were washed twice with 0.15 M NaC and an aliquot of packed RBCs (1.5 ml) was lysed by diluting with 5 volumes of 0.1% formic acid in water, acidified to inhibit further nitroalkylation reactions during sample processing. Samples were centrifuged at 100,000xg for 20 min at 4°C, supernatants collected and GS-  (27). The identity of the GSH-nitroalkene adduct was further confirmed by performing enhanced product ion (EPI) analysis and comparing fragmentation patterns with those obtained from EPI analysis of the synthetic standard GS-OA-NO 2 .
Identification of endogenous nitroalkylated GAPDH in human red blood cells. 300 μl of freshly-obtained, packed RBCs were lysed as before and the cytosolic and membrane fractions separated by ultracentrifugation (100,000xg for 20 min at 4°C). Membranes were resuspended in 100 μl of PBS and aliquots of both cytosolic and membrane fractions (50 μl) resolved by electrophoresis under non-reducing and denaturing conditions (4-15% gradient gel, BioRad, Hercules, CA), then stained with Coomasie Blue. The 36 kDa band corresponding to purified GAPDH was excised and in-gel digestion with trypsin performed. Peptides were eluted and analyzed by nano LC -ESI-MS/MS ((700 nL/min; Finnigan LTQ, Thermo Electron Corporation, CA) using a Zorbax 300SB-C18, 5μm, 0.3 x 50mm column (Agilent Technologies, Chicago).

Crystal Structure of the Rabbit Muscle GAPDH.
The image of GAPDH structure obtained by X-Ray diffraction methods and produced using MolScript and Raster3D was downloaded from Pubmed, Protein Data Bank (1J0X) (28).

RESULTS
The in vitro reaction of LNO 2 with GSH was monitored by ESI-ion trap MS, which revealed a GS-LNO 2 adduct (m/z 631.3), indicative of the nitroalkylation of GSH (Fig. 1A). Either source fragmentation of the m/z 631.3 adduct or residual LNO 2 (m/z 324.2) and GSH (m/z 306.1) accounted for the other ions present in the spectrum.
Collision-induced dissociation (CID) of the GS-LNO 2 adduct precursor ion (m/z 631.3) yielded a main product ion with m/z of 306.1, corresponding to GSH (Fig. 1B). This was further confirmed by MS/MS/MS of the pair m/z 631.3/306.1 (Fig. 1C), which yielded the specific product ions characteristic of GSH (Fig. 1C), as previously reported (29). Similar results were obtained for OA-NO 2 reaction with GSH (not shown).
The nitroalkylation adduct of OA-NO 2 and GSH was also analyzed by reverse phase LC-MS and compared with the elution characteristics of GSH (Fig. 1D). GSH [(M+H) + 308.3] was not retained on column and eluted with the void volume ( Fig. 1D, black tracing). In contrast, the GS-OA-NO 2 adduct [(M+H) + 635.2] eluted at ~74 min, when the solvent gradient reached 38% acetonitrile (Fig. 1D, red tracing). This increased organic phase requirement for elution by reverse phase HPLC reveals that the nitroalkylation of peptides and proteins confers strong hydrophobic character. The elution profile of the GS-OA-NO 2 adduct, revealing multiple resolving peaks (Fig.  1D, red tracing), suggests that different isomers are being formed by the reaction of OA-NO 2 with GSH.
GAPDH, which contains a critical redox and electrophile-sensitive catalytic cysteine (Cys-149, (17)), was incubated with nitro fatty acids. There was a dose-dependent inactivation of GAPDH (0.5 μM) upon incubation of the enzyme with either LNO 2 or OA-NO 2 (0 -10 μM) for 15 min (Fig. 2A). The loss of GAPDH activity as a function of nitroalkene concentration was sigmoidal. The nitroalkene concentration required to inactivate 50% of initial activity (IC 50 ) was ∼3 and ∼4 μM for OA-NO 2 and LNO 2 , respectively. Compared with other recognized biological thiol oxidants, this concentration of nitroalkenes is 30 times less than that of H 2 O 2 and similar to that of ONOOfor inducing similar extents of GAPDH inactivation ( Fig. 2B) (25,30). The time course of OA-NO 2 -mediated GAPDH inactivation was fast, with 50% inactivation occurring within 2 min under the conditions studied (Fig. 2C). The inactivation of GAPDH by OA-NO 2 was strongly pH-dependent, with maximal inactivation obtained at alkaline pH, (Fig. 2D).
The biochemical nature of nitroalkene-GAPDH interactions was investigated in more detail. GAPDH thiol content was determined after OA-NO 2 -mediated enzyme inactivation (Fig. 3A). Nitroalkene-induced GAPDH thiol depletion paralleled the loss of enzyme catalytic activity, with 50% thiol depletion occurring at nitroalkene concentrations inducing a 50% loss of initial enzyme activity (Fig. 3A).
In order to gain further insight into the mechanisms by which nitroalkenes inactivate GAPDH and the oxidation state of the critical thiol, the reversibility of nitroalkene-mediated GAPDH inhibition by other thiol containing reducing agents was studied. The inhibition of GAPDH by nitroalkenes was reversed by incubation with low concentrations of DTT or GSH (Figs. 3B and 3C).
Of significance, inactivation of GAPDH by ONOOand H 2 O 2 was not reversible by DTT or GSH, as previously (25), while both reducing agents restored ∼85% of initial GAPDH activity following OA-NO 2 reaction (Fig. 3B). Extremely low concentrations of thiol (10 μM GSH) reversed GAPDH nitroalkylation and restored ~50% of initial GAPDH activity, emphasizing the reversibility of protein adduction by nitroalkenes under biological conditions (Fig. 3C). In contrast, incubation of nitroalkene-inactivated GAPDH with high concentrations of ascorbic acid (20 mM) did not restore enzymatic activity (not shown).
MALDI-TOF MS analysis of nitroalkenetreated GAPDH showed a shift in the mass of the enzyme (Fig. 4A), revealing covalent modification by nitroalkenes. GAPDH displayed a broader mass distribution following reaction with OA-NO 2 (10 μM, OA-NO 2 /GAPDH molar ratio 20/1), with an increase in mass of up to ~2.0 kDa (∼36 kDa to a maximum of ∼38 kDa; Fig. 4B). This profile of mass shifts indicates that GAPDH was adducted by up to ~ 7 molecules of OA-NO 2 . Reduction of GAPDH-OA-NO 2 adducts with GSH (10 mM) eliminated the OA-NO 2 -induced higher mass species of GAPDH and restored the precursor protein mass (Fig. 4C).
The capacity of OA-NO 2 to form covalent adducts with GAPDH was confirmed by LC-MS using a 2D-linear ion trap. The greater mass resolution of the 2D-linear ion trap resolved the expected mass of native GAPDH (Fig. 4D, black trace) and revealed multiple GAPDH-OA-NO 2 derivatives that all differed by the neutral mass of OA-NO 2 (m/z 327 Da). These nitroalkylated GAPDH derivatives had 3 to 6 OA-NO 2 adducts (Fig. 4D, red trace). A difference of 73 Da between the theoretical mass and the measured mass of GAPDH adducted with 3 OA-NO 2 was observed (Fig. 4D, red trace), suggesting that other modifications (i.e. Cys-, Met-oxidations) were formed. Indeed, Cys and Met were both detected in the native and oxidized form in the tryptic peptide mapping.
To determine precise sites of GAPDH nitroalkylation, native and OA-NO 2 -treated GAPDH were digested with trypsin and analyzed by different mass spectrometric analytical approaches including LC-MS, nanospray LC-MS/MS and MALDI-TOF. Tryptic peptide mass mapping by LC-MS analysis of native GAPDH identified peptides covering 75% of the primary sequence (Supp. Fig. 1 and Supp. Table 1). LC-MS analysis of the peptide map of OA-NO 2treated GAPDH showed marked differences (Table 1 and Fig. 5). There were 5 peptides displaying modified MS and chromatographic behavior in OA-NO 2 -treated GAPDH, while all remaining peptides had similar relative ion intensities and HPLC retention times in both native and treated GAPDH. As an example, peptides #7 and 17, which do not contain Cys or His, are shown to retain similar ion intensity and retention time in OA-NO 2 -treated GAPDH ( Table  1, Fig. 5A). Of the 5 uniquely-behaving peptides, 3 contained His and displayed significantly less relative ion intensity in OA-NO 2 -treated GAPDH (peptides #2, 18 and 23, Table 1 and Fig. 5B). The other two modified peptides contained Cys as the nucleophilic residue and were undetectable in their non-modified form in OA-NO 2 -treated GAPDH. Peptide #19 contains 2 Cys residues, the catalytic active Cys-149 and the Cys-153. The other peptide (#21) contains the Cys-244. (Table  1, Fig. 5C). Of note, the catalytic thiol-containing peptide (#19) contained an intramolecular disulfide bond between both Cys residues in control GAPDH, showing a loss of 2 Da with respect to the expected mass of the peptide (m/z = 1705.86 to 1703.86) ( Table 1 and Fig. 5C). This disulfide was probably formed during trypsin digestion and/or LC-MS analysis, as previously (18). Nanospray LC-MS/MS analysis revealed that His-327 was adducted to OA-NO 2 in this particular peptide ( Fig. 6A and accompanying Table). The MS/MS spectrum and the [M+2H] 2+ ion at m/z 844.34 from this OA-NO 2 -modified peptide are shown ( Fig. 6B and Table 2). The full series of singly-charged y and b fragments were detected, with y and b fragments corresponding to the ions that retain the charge at the carboxyl and amino terminal groups of the peptide, respectively.
The mass difference between y5 and y6, and also between b6 and b7 fragments corresponded to the mass of the His residue (137.06 Da) increased by the neutral mass of OA-NO 2 ( Fig. 6B and Table 2). These results demonstrate that His-327 is the modified residue in this peptide. Modified peptide #18 was also found in analysis of trypsin-digested GAPDH by MALDI-TOF MS (Fig. 6C). Interestingly, postsource decay (PSD) sequencing analysis of this peptide showed a principal, unique product ion with a mass of 437.1 Da.
This fragment corresponds to the immonium ion of His (H = 110.1 Da) adducted to OA-NO 2 (327.3 Da, Figs. 6D and 6E). This adducted immonium ion was also detected by MS/MS analysis performed via nanospray LC-ion trap MS (Fig. 6B). Interestingly, the adducted immonium ion does not appear as a major fragment in this spectrum. This is likely due to differences in the way ions are generated and fragmented in the two different techniques.
The nitroalkylation of peptide #23 (sequence 116-136) by OA-NO 2 was also determined by an increased mass of 327 Da (Supp. Fig. 2). This modification accounted for a 75% decrease in ion intensity of the native peptide upon LC-MS analysis (Fig. 5B). When this modifiedpeptide was analyzed by PSD, the immonium ion of His attached to OA-NO 2 was detected, indicating that His-134 was derivatized by OA-NO 2 in peptide #23 (Supp. Fig. 2). The OA-NO 2adducted immonium ion of His detected by PSD sequencing analysis was a revealing and readilydetected footprint of nitroalkene-modified Hiscontaining peptides by MALDI-TOF MS.  Table), affirming that His-108 is the modified residue in this peptide.
The final peptide detected to have a single OA-NO 2 adduct was peptide #21. The MS/MS spectrum and data for this peptide are shown in Supp. Fig. 4 and the accompanying table. The masses of the b9 fragment and y2* (i.e. y2-NH 3 ), together with the mass difference between the b9 and the b8, demonstrate that Cys-244 is adducted to OA-NO 2 in this peptide.
During nanospray LC-MS/MS, peptide #19, displayed an increase in mass of 654. 6 Da, corresponding to the adduction of two OA-NO 2 . The mass of the fragments b*4 (i.e. b4-NH 3 ) and y7 identifies Cys-149 and Cys-153 as the two modification sites in this peptide (Supp. Fig. 5  The increased hydrophobicity induced by OA-NO 2 adduction of GSH was also manifested by all five OA-NO 2 -modified GAPDH peptides ( Table 2).
The nitroalkylation of peptides profoundly increased the percentage of hydrophobic solvent required to elute adducted peptides from a reverse phase C18-column. Independently of the retention time of the nonmodified, native form of the peptides, all nitroalkylated peptides eluted when the concentration of ACN reached ∼ 40% (Table 2). These results explain why this abundant posttranslational protein modification has not been appreciated sooner, since most LC-MS analyses of peptides rarely utilize ACN concentrations exceeding 30% in gradient elution schemes.
The increased hydrophobicity of nitroalkylated peptides suggests that nitroalkenes may facilitate membrane trafficking following post-translational modification of target proteins. The interaction of nitroalkylated GAPDH with membrane lipids was evaluated by treating GAPDH with increasing concentrations of OA-NO 2 , and then incubating the treated protein with liposomes.
Residual soluble GAPDH was separated from liposome-associated GAPDH by ultracentrifugation and identified by electrophoretic analysis of the supernatant (Fig.  7A). This analysis also revealed a dose-dependent increase in OA-NO 2 -adducted GAPDH association with sedimented liposomes (Fig. 7B). Three of the six GAPDH residues identified to be nitroalkylated in vitro (Cys-149; His-134 and His-327) are located on the surface of the protein and exposed to the solvent (28). The other three potentially-modified residues (His-108; Cys-153 and Cys-244), are buried deeper within the protein (28), suggesting that nitroalkylation at these sites occurs provided that: 1) the initial nitroalkylation of exposed residues induces conformational changes that result in the exposure of previously hidden domains; and/or 2) nitroalkenes are able to diffuse to and react with non-solvent exposed protein residues. These data support that posttranslational nitroalkylation of proteins by nitrated fatty acids will impact protein hydrophobicity, membrane interactions and consequently, subcellular distribution.
To probe whether nitroalkylation occurs in vivo, we analyzed proteins, focusing on GAPDH, from red blood cells obtained from healthy humans.
After separating cytosolic and membrane-associated proteins by electrophoresis under non-reducing conditions, the 36 kDa protein band (which includes GAPDH) was excised and digested in-gel with trypsin. Peptides were eluted and analyzed by nanospray LC-MS/MS (Figs. 8A  and B). The MS/MS spectrum of the human homolog of rabbit GAPDH peptide #19, encompassing the catalytically active Cys-149, showed adduction by OA-NO 2 (Fig. 8A). The mass of the b fragments, particularly fragment b2, showed that Cys-149 was modified by OA-NO 2 in vivo. Another His-containing GAPDH peptide was also OA-NO 2 adducted (Fig. 8B). The increased mass of the peptide by 327 Da and the mass of fragment b3 identified His-303 as the modified, adducted residue. This peptide was found both in cytosolic (not shown) and membrane fractions (Fig. 8B) of red cells, affirming that GAPDH is modified by OA-NO 2 in vivo (Fig. 8).
Finally, due to a) the reversibility of in vitro protein nitroalkylation reactions by GSH, b) the identification of endogenous OA-NO 2 -adducted GAPDH in human red cells and c) the fact that GSH is present in high concentrations in red cells (∼5 mM), the potential presence of nitroalkeneadducted GSH in red cells was examined (Fig. 9). Multiple reaction monitoring (MRM) analysis of partially-purified cytosolic fractions of red cells revealed endogenous species that cochromatographed with synthetic GS-[ 13 C 18 ]OA-NO 2 (Fig. 9A) and GS-[ 13 C 18 ]LNO 2 (Fig. 9B) and displayed identical mass transitions (Figs. 9A and 9B, red vs. black traces).
The identity of endogenous OA-NO 2 and LNO 2 -adducted GSH was further confirmed by enhanced product ion (EPI) analysis and comparison of fragmentation patterns with those obtained from the EPI analysis of synthetic GS-OA-NO 2 (Figs. 10A and 10B). Fragmentation of both the synthetic standard (Fig.  10A) and the endogenous molecules (Fig. 10B) gave the corresponding b2 (m/z 560.3), y2 (m/z 506.3), y2-HNO 2 (m/z 506.3) and Cys-immonium ion (C1), all adducted with OA-NO 2 . To further characterize the GS-OA-NO2 adduct, the y2 fragment of the synthetic adduct was analyzed by MS 3 (Fig. 10C). The CID of this product ion resulted in product ions, including those indicative of the loss of the NO 2 group, supporting that the lipid backbone of the adduct was bonded to the cysteinyl-glycine portion of the molecule and not to the γ-Glu. Moreover, CID of the y2 ion also induced the formation of the cysteinyl immonium ion (C1) adducted to the OA-NO 2 (m/z 403.3). The loss of the NO 2 group from this ion (-HNO 2 ; m/z 356.3) further confirmed that the nitroalkene was attached to the cysteinyl residue of GSH. Based on this fragmentation pattern, it is not possible to determine the location of the NO 2 group on the fatty acid backbone and thus to which carbon the GS is adducted.
The fragmentation profile of the GS-OA-NO 2 adduct strongly depends on the MS mode used to analyze the molecule. In the negative ion mode, collision-induced dissociation (CID) of the GS-LNO 2 adduct precursor ion yielded GSH and LNO 2 as principal product ions (Fig. 1). Interestingly, no ions formed by the combination of GSH with the lipid were observed. In contrast, in the positive ion mode all of the principal product ions (y2, b2, C1) maintained the lipid backbone of the molecule. In aggregate, these results reveal the existence of nitroalkylated GSH in vivo. GS-OA-NO 2 and GS-LNO 2 were present at ∼3.3 and ∼1.3 nM, respectively, in healthy human red cells.

DISCUSSION
Chromatographic and mass spectrometric analyses reveal that nitrated unsaturated fatty acids are potent electrophiles that mediate reversible nitroalkylation reactions with both GSH and the Cys and His residues of proteins. This occurs both in vitro and in vivo (Scheme 1) and is viewed to transduce redox-and NO-dependent cell signaling by a covalent, thiol-reversible post-translational modification that regulates protein structure, function and subcellular distribution.
GAPDH, a tetramer consisting of identical catalytically-active subunits, is a key intermediary metabolism enzyme that reversibly catalyzes the oxidation and phosphorylation of Dglyceraldehyde 3-phosphate (GAP).
Of significance, nitroalkene-induced GAPDH inhibition is reversible by low thiol concentrations, a property which is also not observed with the aforementioned oxidative byproducts. GAPDH not only is inhibited by active site thiol-directed oxidation reactions, but also by the modification of other nucleophilic amino acids (i.e., His, Lys, and non-catalytic Cys residues) (18).
Thus, nitroalkene-induced inhibition of GAPDH could also be due to nitroalkylation of Cys-153, Cys-244 and His-108 and His-327.
While GAPDH inactivation by nitroalkenes was paralleled by a loss of titrateable thiols, the kinetics of enzyme inactivation followed a biphasic, sigmoidal curve (Fig. 2). This supports a differential impact of the multiple documented sites of nitroalkene adduction on enzyme activity, as previously shown for HNE-induced GAPDH inhibition (18).
Recent evidence reveals that GAPDH is a multi-functional protein that displays cell signaling activities beyond its conventionallyviewed role as an intermediary metabolism enzyme. This includes an influence on DNA repair, transcriptional regulation, membrane fusion, tubulin bundling and apoptotic signaling (38). Under physiologic conditions, a significant extent of red cell GAPDH is bound to the membrane of intact cells and is catalytically inactive (39,40). The regulatory mechanisms whereby GAPDH fulfills its non-glycolytic functions and is targeted to different specific intracellular loci are unknown (41), but it is proposed that the functional diversity and differential subcellular distribution of GAPDH is mediated by post-translational modifications and protein-protein and protein-nucleic acid interactions (41).
We reveal herein that nitroalkylation of GAPDH not only directs translocation to the membrane, but also inhibits catalytic activity.
Thus, nitroalkylation of GAPDH provides a mechanism that can explain changes in the subcellular distribution and functional diversity of GAPDH. Future studies should reveal interesting patterns of subcellular distribution and trafficking of not only GAPDH, but also other reversibly nitroalkylated proteins.
GAPDH is a target of ⋅NO, which induces enzyme inhibition (42)(43)(44)(45)(46) and plays an undefined role in promoting the pro-apoptotic translocation of ⋅NO-modified GAPDH into the nucleus and its participation in nuclear events (21). In addition to the present demonstration of nitroalkylation, Cys residues of GAPDH can be S-gluthathionylated (47) and S-nitrosylated (21,44,45). Appreciating that a) ⋅NO-dependent mechanisms mediate fatty acid nitration (5,48) and b) nitrated fatty acids give spurious, false-positive reactions for Snitrosothiols (RSNO) in assays qualitatively and quantitatively identifying this species (2), the contributions of protein nitroalkene derivatives to putative S-nitrosothiol adducts and downstream signaling reactions should be addressed in more detail.
These actions were initially attributed to a capacity to release ⋅NO and to activate PPAR receptors (2,5,9). The present observations reveal a new cell signaling reactivity of nitrated fatty acids -the capability to selectively induce reversible post-translational modification of proteins by nitroalkylation. Current data reveals that nitroalkenes induce pronounced effects on MAPK and JNK cell signaling cascades for reasons only now becoming evident.
For example, protein tyrosine phosphatases contain an active site motif that includes an invariant Cys with a low pK a (53), a property that promotes nucleophilic reactivity and susceptibility to nitroalkylation. Additionally, Cys residues critical for NFkB transcriptional regulation of inflammatory gene expression are potently influenced by nitroalkylation (54).
There is a precedence for GSH forming adducts with modified fatty acids that confer biological activity. The cysteinyl leukotrienes LTC4, LTD4 and LTE4 (GSH S-conjugates of leukotriene A 4 ), recognized by the seventransmembrane-spanning G protein-coupled receptors CysLT 1 and CysLT 2 (55,56), are potent pro-inflammatory lipid mediators. These oxidized fatty acid-GSH adducts are generated via the 5-lipoxygenase pathway and have been implicated in a variety of pathologic conditions (57). It is thus intriguing to speculate whether nitroalkylation of GSH induces signaling actions that parallel those already characterized for nitroalkenes (i.e., generally anti-inflammatory), and whether additional signaling events are mediated by further receptor-ligand interactions.
Nitroalkylation represents a new form of lipid-dependent protein modification, which presently includes co-or post-translational myristoylation, palmitoylation and prenylation reactions (58). The extent and nature of protein nitroalkylation will be dependent on a number of factors summarized in Scheme 2. First, ⋅NOdependent oxidative inflammatory reactions, diet and possible enzymatic synthesis will dictate endogenous levels of free and esterified nitroalkene derivatives (5,48).
Second, the regulated activation of lipases will mediate the hydrolytic release of fatty acid nitroalkene derivatives esterified to complex lipids, thus influencing net levels, anatomic distribution, aqueous vs. hydrophobic partitioning and reactivities of free nitroalkenes. Third, the local environment can lower the pK a and increase the nucleophilicity of critical protein thiol and histidine residues, thus rendering greater reactivity with electrophilic nitroalkenes. Susceptibility to nitroalkylation will also be governed by steric factors and solvent accessibility (59). Fourth, protein nitroalkylation is thiol-reversible, indicating that the net redox status of cells and tissues will also govern extents of biomolecule nitroalkylation.
These aggregate microenvironmental and biochemical properties thus confer a) specificity to nitroalkylation of specific amino acids on particular proteins and b) an ability of cells to regulate extents and sites of protein adduction by nitroalkenes.
In summary, we have described a new reactivity of endogenously-present nitrated fatty acids. These recently-detected derivatives mediate pluripotent signaling activities by acting as a high affinity ligands for PPARγ and PPARα, activating protein kinase signaling cascades and serving as a hydrophobically-stabilized reserve for cGMPdependent ⋅NO signaling. We now show that nitroalkenes mediate the relatively specific, reversible post-translational modification of proteins that serves to transduce redox-and ⋅NOdependent cell signaling by regulating protein function and distribution.  After preincubation of GAPDH (0.5 μM) for 5 min in 50 mM sodium pyrophosphate buffer adjusted to pH 5.5 -10, at 20°C, OA-NO 2 (7.5 μM) was added and after 15 min GAPDH activity was assessed as before. The percentage of control GAPDH activity at each pH was determined.    Table 1. A) Similar relative ion intensities of non-nucleophilic peptides #7 and 17 (m/z 657.3 and 1369.7) were generated by both control and OA-NO 2 -nitroalkylated GAPDH; B) Peptides #2, 18 and 23 containing the nucleophilic, OA-NO 2 -reactive amino acid His were present at lower ion intensities in the tryptic digest of OA-NO 2 -treated GAPDH; and C) Peptides #19 and 21, containing OA-NO 2 -reactive nucleophilic amino acid Cys, were absent in tryptic digests of OA-NO 2 -treated GAPDH.  Table. List of MS/MS fragment ions m/z from peptide #18. Ions that are detected are highlighted in color (Fig. 6B).