In Vitro and in Vivo Protein-bound Tyrosine Nitration Characterized by Diagonal Chromatography^*

Reduction of 3-Nitrotyrosine Peptides by Sodium Dithionite and Analysis by RP-HPLC

The peptide EY(NO₂)QLQYYDPSR (Y(NO₂) = 3-nitrotyrosine) was synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on a 433A peptide synthesizer (Applied Biosystems, Framingham, MA). 2 nmol of this peptide was dissolved in 75 μl of 50 mm borate buffer (pH 9), and the nitro group was reduced by adding 20 μl of freshly prepared 100 mm sodium dithionite. Following 1 min of incubation, the sample was acidified with 10 μl of 10% acetic acid and immediately loaded for RP-HPLC separation onto a 2.1-mm-internal diameter × 150-mm 300SB-C₁₈ column (Zorbax®, Agilent Technologies, Waldbronn, Germany) using an Agilent 1100 Series HPLC system. Following a 10-min wash with 10 mm ammonium acetate (pH 5.5) in water/acetonitrile (98:2, v/v; both Baker HPLC-analyzed, Mallinckrodt Baker B.V., Deventer, The Netherlands), a linear gradient to 10 mm ammonium acetate (pH 5.5) in water/acetonitrile (30:70, v/v) was applied over 100 min at a constant flow rate of 80 μl/min.

Mapping 3-Nitrotyrosines in Tetranitromethane-treated BSA

The COFRADIC sorting protocol was used to map tyrosine residues that were nitrated by in vitro treatment of BSA with tetranitromethane (25). 40 nmol of BSA (Fraction V, ICN Biomedicals Inc., Aurora, OH) was incubated with 80 mm tetranitromethane in 10 mm ammonium bicarbonate buffered at pH 7.8 (total reaction volume was 500 μl). Nitration proceeded for 1 h at 37 °C after which the mixture was acidified with 25 μl of acetic acid. Excess reagents were removed by desalting on a NAP^TM-5 column (Amersham Biosciences), and proteins were collected in 1 ml of 5 mm Tris-HCl (pH 8.7). The volume of this mixture was reduced to 500 μl by vacuum drying after which cysteines were reduced and alkylated with a 25-fold molar excess (over cysteines) of tris(2-carboxyethyl)phosphine (Pierce) and a 50-fold molar excess of iodoacetamide (Sigma-Aldrich) for 30 min at 37 °C. These reagents were then removed by desalting over a NAP-5 column in 1 ml of 10 mm Tris-HCl (pH 8.7). The protein solution was boiled for 10 min and placed on ice for another 10 min. Trypsin (sequencing grade, modified porcine trypsin, Promega Corp., Madison, WI) was then added in a 1:50 (w/w, enzyme/substrate) ratio, and digestion took place overnight at 37 °C and was stopped by adding 50 μl of 10% acetic acid.

One-tenth of the generated peptide mixture (∼4 nmol of BSA) was treated with 0.5% (w/v) H₂O₂ (final concentration) for 30 min at 30 °C. This oxidation step uniformly converts methionines to their sulfoxide derivatives (18), hence preventing that methionyl peptide shift during the secondary separations upon spontaneous oxidation. The so-treated peptide mixture was then separated by RP-HPLC as described above and fractionated into 48 1-min fractions (80 μl each) between 30 and 78 min. To reduce the number of secondary separations, primary fractions that were separated by 16 min were pooled, dried, redissolved, treated with dithionite as indicated above, and separated a second time. Per primary fraction, peptides shifting upon reduction by dithionite were collected in 10-min-wide time intervals starting 13 min before the starting point of collection of each primary fraction. All primary and secondary fractions were sampled by MALDI-MS using an Ultraflex II TOF/TOF mass spectrometer (Bruker Daltonics Inc., Bremen, Germany) operated as described before (26).

In Vitro Nitration of Lysate from Jurkat Cells by Peroxynitrite

200 ml of a Jurkat cell suspension (ATCC, Manassas, VA), at 150 × 10³ cells/ml, was centrifuged for 5 min at 1,500 × g. The pellet was washed twice with 50 ml of ice-cold PBS and lysed with 650 μl of lysis buffer (50 mm Hepes (pH 7.4), 0.9% CHAPS (Sigma-Aldrich), 150 mm NaCl, 1 mm EDTA (Sigma-Aldrich)) and the appropriate amount of a protease inhibitor mixture (Roche Applied Science). Cell lysis was allowed for 10 min on ice followed by centrifugation at 16,000 × g for 30 min at 4 °C to remove cellular debris. 500 μl of this lysate was loaded on a NAP-5 column, and the proteins were collected in 1 ml of 100 mm ammonium bicarbonate buffer at pH 7.8. Protein concentration was determined using the Bradford assay.

Peroxynitrite was freshly prepared as described (27). Briefly, 10 ml of 0.6 m HCl (Sigma-Aldrich) and 0.7 m H₂O₂ (Sigma-Aldrich) was mixed with 10 ml of 0.6 m NaNO₂ and immediately neutralized with 20 ml of 1.5 m NaOH. Excess H₂O₂ was removed by adding 10 mg of MnO₂ for 10 min at 25 °C. To determine the concentration of the peroxynitrite stock solution, the absorbance at 302 nm was measured, and the following formula was used to determine the molar concentration of peroxynitrite: [ONOO⁻] = A₃₀₂/1,670. For nitration of proteins, 25 μl of 40 mm peroxynitrite solution was added for 10 min at 25 °C. Because of the instability of peroxynitrite, this step was repeated once.

Then, 500 μl of the nitrated protein mixture was taken out, and cysteines were reduced with DTT (Sigma-Aldrich; 20 mm final concentration) for 15 min at 37 °C followed by alkylation of cysteines by iodoacetamide (Sigma-Aldrich; 60 mm final concentration) for 30 min at 37 °C. Proteins were then precipitated by adding 9 volumes of ice-cold ethanol and incubation for 4 h at −80 °C followed by centrifugation for 1 h at 100,000 × g (4 °C). The resulting protein pellet was redissolved in 500 μl of 50 mm triethylammonium bicarbonate (pH 7.8) (Sigma-Aldrich). Trypsin was added in a 1:100 ratio (w/w, enzyme/substrate) for overnight digestion at 37 °C.

100 μl of the protein digest was then acidified with 5 μl of 10% acetic acid and treated with 0.5% (w/v) H₂O₂ for 30 min at 30 °C (18). This oxidation step was immediately followed by separation of the peptide mixture by RP-HPLC as described above. The collection of primary fractions, however, was slightly adjusted: peptides eluting between 20 and 80 min were collected into 60 fractions of 1 min (or 80 μl) each. Primary fractions that were separated by 15 min were pooled, resulting in 15 pools of primary fractions. These were then dried in a centrifugal vacuum concentrator and redissolved in 75 μl of 50 mm borate buffer (pH 9.0), and reduction of nitrotyrosines was carried out as described above after which the sample was acidified with 10 μl of 10% acetic acid and immediately separated by RP-HPLC under identical conditions as during the primary run. 3-Aminotyrosine-containing peptides were collected in six equal volume fractions from 12 to 2 min prior to the collection interval of each primary fraction. A total of 360 secondary fractions were in this way collected, and to reduce mass spectrometer analysis time, secondary fractions that were separated by 15 min were repooled, resulting in 90 fractions for further analysis.

Identification of Tyrosine Nitration Sites in Serum Proteins of Sepsis Mouse Model

Female C57BL6/J mice were from Janvier (Le Genest-St. Isle, France) and used at the age of 8–10 weeks. The mice were maintained in a temperature-controlled, air-conditioned specific pathogen-free animal facility with 14/10-h light/dark cycles and received food and water ad libitum. All experiments were approved by the animal ethics committee of the Faculty of Sciences of Ghent University (Belgium) and performed according to its guidelines.

Pathogenic Salmonella enteritidis (serovar typhimurium) were from ATCC. Bacteria were diluted in lipopolysaccharide-free PBS to a concentration of 4·10⁷ colony-forming units/ml, and 0.25 ml was intravenously injected per mouse in the right tail vein. Mice were bled daily at 9 a.m. at the retro-orbital plexus. Blood was allowed to clot for 60 min at 37 °C and further overnight at 4 °C. The clot was removed, and cells were pelleted by centrifugation at 14,000 rpm for 15 min. Serum was separated and stored at −20 °C until further use. Five serum samples taken at days 4 and 5 were pooled to obtain a total volume of 150 μl of crude serum followed by depletion of the top three most abundant proteins: albumin, immunoglobulin G, and transferrin. A commercially available affinity system was used for this purpose (Multiple Affinity Removal System, Agilent Technologies). 2 ml of the depleted serum was reduced to 900 μl by vacuum drying. The pH of the solution was adjusted to 8.7 by addition of 100 μl of 1 m Tris·HCl (pH 8.7). Alkylation of cysteines was carried out using final concentrations of 60 mm iodoacetamide and 30 mm tris(2-carboxyethyl)phosphine for 30 min at 37 °C. Excess reagents were removed by desalting over a NAP-10 (Amersham Biosciences) desalting column in 1.5 ml of 50 mm triethylammonium bicarbonate buffer (pH 7.8). The protein concentration was determined using the Bradford assay to be 0.86 mg/ml. Proteins were heated prior to digestion for 10 min at 70 °C, and after cooling down on ice, trypsin was added in a 1:100 ratio (w/w) followed by digestion overnight at 37 °C. The volume of the peptide mixture was reduced to 300 μl by vacuum drying after which 100 μl of this mixture was acidified with 5 μl of 10% acetic acid, and methionines were oxidized as outlined above followed by immediate injection onto the RP-HPLC column for COFRADIC sorting as described above.

LC-MS/MS Analysis of Jurkat Peptides

Each secondary fraction was redissolved in 20 μl of 0.1% formic acid in water (solvent A). 5 μl of this peptide mixture was used for nanoflow-LC tandem mass spectrometry on an Agilent 1200 HPLC system coupled to a 7-tesla LTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany). Here a trapping column (Zorbax, 300SB-C₁₈, 5-μm particles, 5-mm length × 300-μm diameter (Agilent Technologies)) was connected to an analytical column (Zorbax, 300SB-C₁₈, 3.5-μm particles, 150-mm length × 75-μm diameter (Agilent Technologies)). Peptides were loaded at 4 μl/min on the trapping column for 10 min, and following flow splitting to 300 nl/min, peptides were transferred to the analytical column and eluted by a gradient of acetonitrile (a 1.5% solvent B increase per minute; solvent B was 80% acetonitrile in 0.1% formic acid). The eluent was sprayed via emitter tips (FS360-20-10-N5-C12, New Objective, MS Wil GmbH, Wil, Switzerland) butt-connected to the analytical column.

The LTQ-FT mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the three most abundant peaks in a given MS spectrum. Full-scan MS spectra were acquired in the FT-ICR cell at a target value of 1e5 with a resolution of 100,000. The three most intense ions were then isolated for fragmentation in the linear ion trap. In this LTQ, MS/MS scans were recorded in profile mode at a target value of 10,000. Peptides were fragmented after filling the ion trap with a maximum ion time of 100 ms and a maximum of 1e4 ion counts. From the MS/MS data in each LC-MS/MS run, Mascot generic files (mgf) were created using the Mascot Distiller software (version 2.2.1.0, Matrix Science Ltd., London, UK). When generating peak lists, grouping of spectra was performed with a maximum intermediate retention time of 30 s and maximum intermediate scan count of 5. Grouping was further done with 0.1-Da precursor ion tolerance. A peak list was only generated when the spectrum contained more than 10 peaks. There was no deisotoping, and the relative signal to noise limit for both precursor ions and fragment ions was set to 2. These peak lists were then searched with Mascot using the Mascot Daemon interface (version 2.2.0, Matrix Science Ltd.). The Mascot search parameters were as follows. Searches were performed in the Swiss-Prot database with taxonomy set to human (database version 56.4; 20,328 human protein sequences). Methionine oxidation to its sulfoxide was set as a fixed modification. Variable modifications were 3-aminotyrosine, sulfation of 3-aminotyrosine, acetylation of protein N termini, carbamidomethylation of cysteine, and oxidation of the carbamidomethylated cysteine. Trypsin was set as the enzyme used (one missed cleavage was allowed), the mass tolerance on the precursor ion was set to 10 ppm, and the mass tolerance on the fragment ions was set to 0.5 Da. In addition, the C13 setting of Mascot was set to 1. For the estimation of the false discovery rate of peptides identified by Mascot, searches were performed in a concatenated decoy database consisting of a reversed Swiss-Prot database (28) at the 95% confidence level.

LC-MS/MS Analysis of Mouse Serum Peptides

Secondary fractions were redissolved in 15 μl of 2% acetonitrile, and 8 μl was used for LC-MS/MS analysis using an Ultimate 3000 HPLC system (Dionex, Amsterdam, The Netherlands) in line connected to an LTQ Orbitrap XL mass spectrometer (Thermo Electron). Peptides were first trapped on a trapping column (PepMap^TM C₁₈ column, 0.3-mm inner diameter × 5 mm (Dionex)), and following back-flushing from the trapping column, the sample was loaded on a 75-μm-inner diameter × 150-mm reverse-phase column (PepMap C₁₈, Dionex). Peptides were eluted with a linear gradient of a 1.8% solvent B′ (0.05% formic acid in water/acetonitrile (2:8, v/v)) increase per minute at a constant flow rate of 300 nl/min.

The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the six most abundant ion peaks per MS spectrum. Full-scan MS spectra were acquired at a target value of 1e6 with a resolution of 30,000. The six most intense ions were then isolated for fragmentation in the linear ion trap. In the LTQ, MS/MS scans were recorded in profile mode at a target value of 5,000. Peptides were fragmented after filling the ion trap with a maximum ion time of 10 ms and a maximum of 1e4 ion counts. From the MS/MS data in each LC-run, Mascot generic files (mgf) were created using the Mascot Distiller software (version 2.2.1.0, Matrix Science Ltd.). When generating these peak lists, grouping of spectra was performed with a maximum intermediate retention time of 30 s and maximum intermediate scan count of 5 used where possible. Grouping was done with 0.1-Da tolerance on the precursor ion. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks, no deisotoping was performed, and the relative signal to noise limit was set at 2.

Such generated peak lists were then searched with Mascot using the Mascot Daemon interface (version 2.2.0, Matrix Science Ltd.). Spectra were searched against the Swiss-Prot database, and taxonomy was set to Mus musculus (database version 56.4; 15,988 mouse protein sequences). Variable modifications were set to methionine oxidation, pyroglutamate formation of N-terminal glutamine, carbamidomethylation of cysteines, oxidation of the carbamidomethylated cysteine, acetylation of the N terminus, and deamidation of glutamine or asparagines. Mass tolerance of the precursor ions was set to 10 ppm, and mass tolerance of the fragment ions was set to 0.5 Da. The peptide charge was set to 1+, 2+, or 3+, and one missed tryptic cleavage site was allowed. Also, the C13 setting of Mascot was to 1. Only peptides that were ranked 1 and scored above the threshold score set at 95% confidence were withheld. The FDR was estimated as indicated above for the LTQ data.

Quality Assessment of Mascot-identified Tyrosine-nitrated Peptides Using Peptizer Algorithm

Following Mascot database searching of the Jurkat MS/MS data, 683 MS/MS spectra were identified to belong to peptides containing 3-aminotyrosine or sulfated 3-aminotyrosine. To assess the quality of these peptide identifications and thus reduce the number of potential false positive identifications, the following quality assumptions were further inspected by Peptizer (29). The peptide identification scored more than 10 units above the 95% identity threshold, the peptide was longer than 8 amino acids, only a single peptide was allowed to score above the 95% identity threshold, at least 30% of b ions and y ions were observed, and an (optional) MS/MS-unstable proline peptide bond produced dominant b ions or y ions. If two or more of the above assumptions were not fulfilled by a suggested identified peptide, this peptide was not included in the high quality list. Most importantly, Peptizer also verified that the actual modified tyrosine residue was flanked on both sides by b, b²⁺, y, or y²⁺ fragment ions before including peptides in the here reported high quality list. The Peptizer profile used is available upon request. Supplemental Table 1 lists the identifications that passed the Peptizer criteria (note that whenever a peptide was linked to several protein sequences stored in the database this is indicated in the table). The supplemental information lists the identified MS/MS spectra from both the Jurkat and the mouse serum study.

Diagonal Reverse-phase Chromatographic Isolation of 3-Nitrotyrosine Peptides after Reduction to 3-Aminotyrosine Peptides by Na₂S₂O₄

A previous report suggests that 3-aminotyrosine peptides are more hydrophilic than their nitrated counterparts (30). Here this is illustrated by the UV absorbance chromatograms shown in Fig. 1: the 3-nitrotyrosine peptide eluted at about 48 min (solid trace), and the 3-aminotyrosine peptide generated by dithionite reduction was more hydrophilic, eluting 6 min earlier (dotted trace). As stated before (18, 31), the extent of such evoked chromatographic shifts ideally must be sufficiently large to reduce overlap between sorted peptides and peptides unaffected by the COFRADIC sorting reaction. Although the extent of the hydrophilic shift may depend on the sequence of the peptide, using the RP-HPLC setup described above, the evoked shift was typically more than 4 min, implying sufficient separation between sorted (3-aminotyrosyl) and unsorted peptides. In general, the average yield of the reduction of the nitrotyrosine was over 95% as judged by the drop of the UV absorbance intensity of the nitrated peptide (Fig. 1).

Identification of Tyrosine Nitration Sites in Tetranitromethane-treated BSA

In a second study, BSA was in vitro nitrated and digested with trypsin, and the resulting peptide mixture was separated by RP-HPLC (Fig. 2A). Primary fraction 32 contained a peptide ion with a mass of 1,612.774 Da (Fig. 2C), corresponding to the expected mass of singly nitrated DAFLGSFLYEYSR, a tryptic peptide spanning residues 347–359 of BSA. Characteristic satellite peaks 16, 30, and 32 Da lower than the mass of the intact peptide ion were previously observed in MALDI-MS spectra of 3-nitrotyrosyl peptides (25, 32) and confirm tyrosine nitration of this peptide. Upon treatment with dithionite and reseparation by RP-HPLC (Fig. 2B), a peptide ion with a mass of 1,582.653 Da appears in the MALDI-MS spectrum of the secondary fraction containing the sorted peptide (Fig. 2D). This mass can be linked to DAFLGSFLYEYSR, now with one 3-aminotyrosine residue. MALDI-PSD analysis further confirmed the nature of this peptide and indicated that tyrosine 357 was indeed present in its amino form and was thus in vitro nitrated by tetranitromethane (Fig. 2E). Interestingly, tyrosine 355 was not nitrated, although it is only two residues distant from the nitrated tyrosine on position 357.

Secreted BSA contains 20 tyrosines, and following MALDI-MS analysis of all secondary fractions and MALDI-PSD analysis on sorted peptides, we identified four tyrosines that were nitrated by tetranitromethane: tyrosines 161, 357, 364, and 424. Interestingly, these tyrosines meet nitration criteria that were previously set: all are surface-accessible and have negatively charged amino acids but no cysteines nearby (33). We could not show nitration of other tyrosines, which may be due to several reasons such as the fact that they fail to meet in vitro protein nitration criteria or they reside in peptides that are not analyzable by LC-MS/MS.

Protein-bound in Vitro Tyrosine Nitration in Jurkat Cell Lysate

We then isolated and identified 3-nitrotyrosine-containing peptides in a much more complex mixture, a Jurkat cell lysate treated with peroxynitrite. Because earlier reports (13, 34) showed that reduction of 3-nitrotyrosine by sodium dithionite may lead to two end products, 3-aminotyrosine and sulfated 3-aminotyrosine (probably caused by the addition of an SO₃H group onto the ortho position of the aromatic benzene ring), we further accounted for sulfation of 3-aminotyrosine side chains during database searching. Using 300 μg of starting material for the COFRADIC procedure, we initially identified 683 LTQ-FT MS/MS spectra belonging to 443 unique sequences from 342 different proteins. Recently Stevens et al. (14) reported several factors that lead to misidentification of 3-nitrotyrosine-containing peptides. Therefore, we introduced two approaches to create a high quality list of the identified spectra and thus filter out possible false positive identifications. First, we estimated the FDR of Mascot identifications using a concatenated decoy database search, resulting in an FDR value of 2.6%. Next, we used the in-house developed Peptizer program (29) to filter out all peptides that did not meet strict criteria such as the presence of flanking b and y fragment ions at the 3-aminotyrosine residue (or its sulfated counterpart). The result is a high quality list of 491 LTQ-FT MS/MS spectra that were assigned to 335 different 3-nitrotyrosine peptides from 267 unique proteins (see supplemental Table 1). Importantly, Peptizer not only removed all spectra linked to 3-aminotyrosine-containing peptides identified in the decoy database but also removed peptides identified in the “forward” database that were highly likely to be false. An example of the latter is the N-terminal peptide of fructose-bisphosphate aldolase 1. Interestingly, the murine N-terminal aldolase peptide (nitrated on Tyr⁵) was identified in another study (10), and here we initially identified its human counterpart (nitrated on Tyr³ and Tyr⁵), but Peptizer removed it from the list of identifications as it did not meet the criteria of Peptizer and was therefore considered as a possible false positive identification.

Our list represents a map of Jurkat tyrosines that are targeted by the peroxynitrite radical, and among the 335 different nitrated sequences, we found previously identified nitration sites (both from in vitro as in vivo experiments) such as Tyr¹⁴⁸ in the sequence ITLDNAY*MEK (where Y* is 3-aminotyrosine) from pyruvate kinase (see Fig. 3A), which was previously identified in skeletal muscle tissue from 34-month-old Fisher 344/Brown Norway F1 rats (35). Interestingly, we also picked up its sulfated form, and Fig. 3B clearly shows an 80-Da increase on the peptide fragment ions carrying the sulfated 3-aminotyrosine. 75.6% of the reduced nitrated sequences (371 of 491 spectra) were actually identified to carry sulfated 3-aminotyrosine. On the peptide level, this means that 51 peptides (∼15%) were identified in their non-sulfated form, 45 peptides (∼13%) were identified in both forms, and the majority (239 peptides or ∼71%) were identified as sulfated 3-aminotyrosine peptides, which illustrates the importance of accounting for this modification in database searches. Using the precursor ion intensities of peptides that were found both sulfated and non-sulfated, we estimated that the degree of sulfation ranged from 5 to 90%, again showing that sulfation of aminotyrosine residues is an important artifact when dithionite is used. On the other hand, however, we currently do not hold conclusive proof on how aminotyrosine residues become sulfated in our system but noticed that by increasing the excess of dithionite in the reduction reaction the degree of sulfation was also increased, hinting to a role of dithionite (or its oxidized products) in this sulfation reaction.

Looking more into detail in the list of identified peptides, Tyr¹⁶¹ and Tyr³⁵⁷ of the tubulin α-1 β chain were here identified as nitrated. Both tyrosines were found by Tedeschi et al. (36) to play an important role during nerve growth factor-induced neuronal differentiation. In addition, nitration of Tyr²²⁴ found here was previously described by Fiore et al. (37), specifically during grade IV human glioma. Haqqani et al. (38) reported histone nitration, and we confirmed Tyr⁷² of histone H4 and Tyr⁴³ of histone H2B as nitration spots upon in vitro peroxynitrite treatment.

Thus, because tyrosine nitration sites previously found in vivo were also identified in our study, it is tempting to state that in vitro nitration by peroxynitrite does not target tyrosines randomly but might well resemble physiological nitration found in living cells. Hence, we asked whether sequence or structural features could help explain preferred sites of peroxynitrite nitration of tyrosines. It is believed that nitration targets reside in specific protein segments containing negatively charged or turn-inducing amino acids (33, 39). At first sight, many of the peptides we identified appeared to contain such amino acids; however, using the Two Sample Logo tool (40), we found no absolute preferences of specific amino acids surrounding the tyrosine nitration site (supplemental Fig. 1A), an observation that was also made by Souza et al. (5). This possibly points to the local three-dimensional protein structure as an important determinant for tyrosine nitration rather than the primary protein sequence. Considering secondary structure, a slight increase of nitration sites in coiled regions is observed (supplemental Fig. 1B).

Several reports also point to a dynamic interplay between protein tyrosine nitration and phosphorylation (41, 42). 22 identified peptides (about 6.5%) are modified on tyrosines that are known to be phosphorylated by a wide diversity of tyrosine kinases. This, combined with the chance that a random tyrosine-containing peptide is phosphorylated (0.46%), indicates that our list contains a tendency toward tyrosines that are phosphorylated or indicates that nitration, similar to phosphorylation, occurs on tyrosine residues that are surface-accessible. Note that the value of 0.46% was calculated by dividing the number of experimentally found tyrosine phosphorylation sites in human proteins (1,387 sites stored in Phospho.ELM) by the number of tyrosines found in human proteins (299,816 found in Swiss-Prot).

Tyrosine Nitration of Serum Proteins during Salmonella-induced Septic Shock

Intravenous administration of Salmonella into mice causes severe septic shock and leads to oxidative burst (43). As a result, oxygen- and nitrogen-derived radicals are produced to create a toxic environment for the invading pathogen. The excess radicals produced are scavenged by serum proteins and cause modifications such as nitration of tyrosines.

Using our COFRADIC approach, we investigated whether we could identify nitrated tyrosine residues in serum proteins of mice infected with Salmonella. Here we linked 28 MS/MS spectra to peptides potentially containing in vivo nitrated tyrosines. The spectra were then filtered by Peptizer to eliminate possible false positive spectra using the same agents as described above. Finally, eight MS/MS spectra were withheld pointing to six different peptides from four unique serum proteins (Table I).

Three sites in α₂-macroglobulin were identified: tyrosines 1019, 1024, and 1330. Earlier reports indeed indicated that α₂-macroglobulin was susceptible for nitration (44) and thus probably serves as a scavenger for excess radical formation. Apolipoprotein A-I is also known to be nitrated during conditions of oxidative stress because of its close association with myeloperoxidase (45–48), and Tyr⁴³ of this protein was found nitrated. The study of Parastatidis et al. (47) clearly showed that apolipoprotein A-I serves as a protector protein that scavenges radicals and controls the oxidative burden. Tyr⁶⁶ from haptoglobin was also nitrated in the sepsis model, and nitration of haptoglobin is known to influence its function. Finally, we identified Tyr⁴¹⁰ from the vitamin D-binding protein as a nitration site during sepsis.