Nitroproteins Identified in Human Ex-smoker Bronchoalveolar Lavage Fluid

Abstract

The long-term goal of our study is to identify chronic obstructive pulmonary disease (COPD)-related bronchoalveolar lavage fluid (BALF) nitroproteins to clarify COPD pathological mechanisms and to discover biomarkers of COPD. The goal of the present study was to detect the presence of, and potential roles of, nitroproteins in, human ex-smoker (without COPD) BALF samples. Nitroproteins were immunoprecipitated from two separate BALF samples, and digested with trypsin; and tryptic peptides were analyzed with matrix-assisted laser desorption/ionization (MALDI)-tandem mass spectrometry (MS/MS). Each MS/MS spectrum was composed of accumulated scans (n = 50–100). The MS/MS data were searched with BioWorks 2.0 TuboSequest in the SwissProt database to generate the amino acid sequence, which was evaluated manually. Eleven nitrotyrosine sites were identified in eight proteins, including progestin and adipoQ receptor family member III, zinc finger protein 432, proteasome subunit alpha type 2, NADH-ubiquinone oxidoreductase B14, slit homolog 1 protein, lysozyme, aldose 1-epimerase, and PTS system lactose-specific EIICB component. Each nitrotyrosine site was located within a specific protein domain and motif. Those identified nitrated proteins could be involved in multiple functional metabolic systems, including transcriptional regulation, mitochondrial complex, immune system, and energy metabolism.

Cigarette smoke, a major cause of inflammatory-related lung diseases such as chronic obstructive pulmonary disease (COPD), contains a variety of oxidants and nitrosants such as nitric oxide (NO) [1] that could initiate the inflammatory response to airway and lung insult. Alveolar macrophages, neutrophils, CD8⁺ lymphocytes, other inflammatory cells, and airway epithelial cells generate and release reactive oxygen species (ROS) such as the superoxide anion (O₂^.−), hydrogen peroxide (H₂O₂), and the hydroxyl radical (OH·) through an NADPH-oxidase-dependent mechanism; and the nitric oxide (NO·) free radical through a nitric oxide synthase (NOS)-dependent mechanism. Inducible (iNOS) can generate large amounts of NO for an extended period of time during an inflammatory response compared to neuronal (nNOS) and endothelial NOS (eNOS). Most of the cellular toxic effects of NO result from its rapid reaction with O₂^.− to form the strong nitrosant peroxynitrite anion (ONOO⁻)[2, 3]. Peroxynitrite causes the nitration (addition of –NO₂) of a protein tyrosine residue to provoke inhibition of mitochondria respiration, protein dysfunction, and damage to cell membranes [4, 5] to involve the pathophysiological processes of inflammatory-related lung diseases. Tyrosine nitration is also possibly generated through heme peroxidase (such as myeloperoxidase, MPO; and xanthine oxydase, XO)-dependent mechanisms [4].

Studies have shown that oxidants and nitrosants such as peroxynitrite in the gas phase of cigarette smoke can pass through the pulmonary alveolar wall into blood to induce systemic oxidative stress and to increase the serum level of 3-nitrotyrosine [6]. The level of 3-nitrotyrosine in a smoker is significantly higher than in a non-smoker [7]. Tobacco smoke impairs the normal process of NO generation to alter the function of peripheral muscle [8]. Tobacco nicotine significantly increased superoxide anions and 3-nitrotyrosine-positive proteins, and exogenous peroxynitrite mimicked the effects of nicotine on the AMP-activated protein kinase (AMPK) that acts as a major energy sensor and regulator in adipose tissues [9], and the tyrosine nitration of sarcoplasmic-endoplasmic reticulum Ca²⁺ adenosine triphosphatase 2 (SERCA2) in COPD patients could contribute to their low body mass index (BMI) [10]. Chronic smoking causes the decreased exhaled NO and an increased level of nitrotyrosine [11]. Cigarette smoke-mediated oxidative stress induced an inflammatory response in the lungs by stimulating the release of proinflammatory cytokines such as interleukin 8 (IL-8) via the activation of NF-kB [12]. Smoking can cause nitration of a protein tyrosine residue [13]. Antioxidants such as superoxide dismutase can protect smoke-induced inflammation and conteract the proteolytic cascade that leads to emphysema formation [14].

Protein nitration adds 45 mass units (NO₂ – H) to a tyrosine residue [15]. Tandem mass spectrometry (MS/MS) enables one to monitor that mass shift, obtain an amino acid sequence of a nitropeptide, and identify the low-abundance nitrotyrosine sites (∼ 1 in 10⁶ tyrosines [16, 17]) after a nitrotyrosine antibody-based immunoaffinity preferential enrichment of endogenous nitrotyrosine-containing proteins [18, 19].

The goal of the present study was to detect the presence of, and the potential roles of, nitroproteins in human ex-smoker (without COPD) BALF samples. Nitroproteins in BALF were preferentially selected with immunoprecipitation (IP). Nitrotyrosine sites were identified with tandem mass spectrometry. Each nitrotyrosine site was located within a specific protein domain and motif. Those nitroproteins could be involved in multiple functional metabolic systems, including transcriptional regulation, mitochondrial complex, immune system, and energy metabolism; which provides new insights into the mechanisms of oxidative/nitrative stress in cigarette smoke-related lung diseases and new clues to biomarker discovery.

MATERIALS AND METHODS

Human BALF collection

The protocol of collecting human BALF was approved by the Institutional Review Board at Chiesi Pharmaceutical Company (Parma, Italy). Written informed consent was obtained from each subject to enroll in the study. Briefly, BAL was performed by professional clinicians. Five separate 30 ml aliquots of 0.9% sterile saline were instilled into the right middle lobe or lingula. BALF samples were collected, aliquoted into polypropylene tubes, and stored at −80 °C prior to further analyses. A 5-ml aliquot of BALF on dry-ice was shipped by air to the University of Tennessee Health Science Center in Memphis for nitroproteomics analysis.

BALF protein preparation

Two separate 5-ml BALF samples (male, ex-smoker without COPD, 56 and 66 year-old) were vacuum-dried (0°C), and the residues were redissolved in 180μl of Pierce M-PER protein extraction buffer; the solutions were centrifuged (15,000 x g; 30 min). The supernatants of the two samples were combined (∼ 350 μl) for nitrotyrosine immunoaffinity analysis.

Nitroprotein immunoprecipitation

Nitrotyrosine immunoprecipitations were performed as described previously [19]. Briefly, rabbit anti-nitrotyrosine polyclonal antibody (92 μg, Chemicon International, Temecula, CA, USA) was coupled with immunopure immobilized protein G beads (400 μl, Pierce) for 2h with gentle shaking in binding buffer (0.14M NaCl, 0.008M sodium phosphate, 0.002M potassium phosphate, and 0.01M KCl; pH 7.4), and washed to remove any unbound antibody (Pierce Immunoprecipitation Kit, product 45225). The bound antibody was covalently crosslinked to protein G with gentle shaking (40 min) in disuccinimidyl suberate (final concentration 0.0025%); the beads were washed briefly with a low pH buffer (pH 2.8) to remove non-crosslinked antibody, and re-equilibrated into the above binding buffer. The immobilized anti-nitrotyrosine antibody (∼92 μg) was incubated (4 °C, overnight) with the prepared BALF protein samples to capture the nitrotyrosine-containing proteins; the column was washed (3x) with the binding buffer (400 μl), and nitroproteins were eluted with Pierce Elution buffer into a volume of ∼250 μL.

Tryptic peptide preparation

An aliquot (20 μL) of IP products was treated with 1 μL of 1 M Tris (pH 9.5). Sequencing grade-modified trypsin (20 μg, Promega, Madison, WI, USA) was dissolved in a volume (100 μl, pH 8.2) of solution that consisted of 5 μl trypsin resuspension buffer that contained 50 mM acetic acid (pH 2.8) and 95 μl of 200 mM NH₄HCO₃ (pH 8.2). The enzyme digestion reaction system (100 μl, final concentration of NH₄HCO₃ = 50 mM, pH8.1), which consisted of 21 μl neutralized IP products, 25 μl trypsin solution, and 54 μl ddH₂O, was incubated (overnight; 37 °C). An aliquot (20 μL) of the tryptic peptide mixture was purified with a Millipore ZipTip C18 column according to the manufacturer’s instructions (Millipore, Billerica, MA, USA). The purified tryptic peptides were eluted directly from the microcolumn onto a vMALDI plate with 2 μL of an α-cyano-4-hydroxycinnamic acid (CHCA) solution (2.5 mg/ml CHCA in 50 % v/v acetonitrile/0.1 % v/v trifluoroacetic acid); that solution was dried in ambient air for mass spectrometry (MS) analysis.

Protein identification with MALDI-tandem mass spectrometry

The tryptic peptides were analyzed with a vMALDI-LTQ tandem mass spectrometer in the “Nth-order double play” data-dependent experimental mode to obtain the amino acid sequence of each peptide [19]. Briefly, the crystal-positioning system (CPS) and auto spectrum filter (ASF; threshold = 500 counts for an MS scan and 250 counts for an MS² scan) were enabled for the vMALDI source. The automatic gain control (AGC) was enabled to automatically adjust the number of laser shots to maintain the quality of the spectra. For an MS scan, high-mass range (m/z 600–4000), normal scan rate, full scan, polarity, profile data type, and five microscans of each experiment were used. For an MS² scan, the 50 most-intense peaks in the full MS spectrum, high-mass range (m/z 50–4000), normal scan rate, polarity, centriod data type, isolation width 3.0 Th, normalized collision energy 40 (arbitrary units), default charge state 1, minimal signal threshold 100 counts, activation Q value 0.25, activation time 30 ms, and five microscans of each experiment were used. Instrument operation and data acquisition utilized the Xcalibur software package (ThermoFinnigan). Initial protein identifications from MS/MS data utilized the Bioworks TurboSequest software search engine (ThermoFinnigan, version 3.2) and the Swiss-Prot protein database. The Swiss-Prot database search parameters included 2 allowed missed tryptic cleavage sites, precursor-ion mass tolerance = 2 Da, fragmention mass tolerance = 1.0 Da, and protein modifications for Tyr nitration (+ 45 mass unit), Asn and Gln deamidation (+ 1 mass unit), and Met oxidation (+ 16 mass unit). Each nitrotyrosine-containing peptide was examined manually, as described previously [19]. The residue that preceded the N-terminus or C-terminus must be K or R with singly charged b-, y-, and a-ion series for a nitrotyrosine-positive search result. An accumulated MS² spectrum (n = 50–100 scans) of each nitrotyrosine-positive search result was acquired on the vMALDI-LTQ Tune page to improve the signal-to-noise (S/N) ratio, and was used to search the Swiss-Prot database with Bioworks 3.2 to corroborate each positive search result.

Furthermore, most of the prominent peaks within the MS spectra were selected to directly acquire the accumulated (n = 50) MS² spectrum whether it was a nitropeptide or not. The MS/MS data were used to identify the protein and nitrotyrosine-site as described above. NCBI BLASTP (version 2.2.17; http://ca.expasy.org/cgi-bin/blast.pl) was used to determine the identity of each nitropetide that was aligned to a protein in the UniProtKB Database (6,489K sequences that consist of Swiss-Prot and TrEMBL databases; July 1, 2008).

Protein domain and pathway analyses

Protein domain and motif analyses were carried out with ScanProsite (http://us.expasy.org/tools/scanprosite/), and Motifscan (http://myhits.isb-sib.ch/cgi-bin/motif_scan). Each nitrotyrosine-site was located within the corresponding protein domain and motif. The pathway networks that could involve the nitroproteins were analyzed with Ingenuity Pathways Analysis software (Ingenuity® Systems, www.ingenuity.com).

RESULTS

Nitroproteins identified in BALF

The tryptic peptides were subjected to vMALDI MS/MS analysis, after nitroprotein immunoaffinity enrichment and digestion with trypsin. MS/MS data provided the amino acid sequences of nine nitrotyrosine-containing peptides from human ex-smoker BALF (Table 1; Supplemental Figures A–H). Each peptide amino acid sequence and nitrotyrosine-site was confirmed with a manual analysis of the MS/MS spectrum, including spectral quality and the interpretation of the b- and y-ion series. Furthermore, each nitropeptide was also confirmed with accumulated MS/MS spectra (n = 50–100). The amino acid sequence of each nitropeptide was exactly matched to a protein with Bioworks TuboSequest analysis in the Swiss-Prot protein database: progestin and adipoQ receptor family member III, zinc finger protein 432, proteasome subunit alpha type 2, NADH-ubiquinone oxidoreductase B14, slit homolog 1 protein, lysozyme, aldose 1-epimerase, and PTS system lactose-specific EIICB component. Table 1 contains those nitroproteins: protein name, species, Swiss-Prot database accession number, nitropeptide amino acid sequence, nitrotyrosine site, singly-charged precursor ion ([M + H]⁺), and TuboSequest Score.

Table 1.

Nitroproteins Identified in Human Ex-smoker BALF

Protein Name (Species)	Swiss-Prot No.	Nitrotyrosine-containing peptide	Nitrotyrosine site	[M +H]+	Sequest Score	E-value (NCBI 6489K)	Protein domain and motif containing nitrotyrosine	Protein Function
Progestin and adipoQ receptor family member III (Human)	Q6TCH7	R.LYTY*EQIPGSLKD NPYITDGYRAYLPSR. L	Y33	3338.6	3.53	3.0E-19	Cytoplasmic	Multi-pass membrane protein
Zinc finger protein 432 (Human)	O94892	K.DLYRDVMLEIY*SN LLSMGYQVSKPDALS K.L	Y41	3393.7	3.04	7.0E-20	KRAB domain	Transcriptional regulation
Proteasome subunit alpha type 2 (Human)	P25787	K.DY*LAAIA.-	Y229	781.4	1.61	4.6E+00	Functional chain	Multicatalytic proteinase complex with an ATP-dependent proteolytic activity
NADH-ubiquinone oxidoreductase B14 subunit (Human)	P56556	R.ELY*RAWY*REVP NTVHQFQ#LDITVK.M	Y36, Y40	3096.6	2.91	5.0E-16	Functional chain, component of mitochondrial Complex I	Accesory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase
Slit homolog 1 protein (Rat)	O88279	K.HDCVN#GGVCVDG IGNYTCQCPLQY*TG R.A	Y1035	2918.2	2.74	1.0E-18	EGF-like domain 3	Secreted; Act as molecular guidance cue in cellular migration.
Lysozyme (ANOGA)	Q17005	K.NKNGSTDYGIFQIN NKY*WCDSGYGSN#D CK.I	Y79	3337.4	2.08	9.0E-21	Tyrosine Kinase phosphorylation motif	Have primarily a bacteriolytic function; associated with enhance the activity of immunoagents.
		K.NKNGSTDYGIFQIN N#KYWCDSGY*GSND CK.I	Y85	3337.4	2.08	9.0E-21
Aldose 1-epimerase (ACICA)	P05149	K.FSLDGKTY*NLEKN NGPN#SLHSGN#PGFD K.R	Y118	3097.5	3.77	6.0E-18	Tyrosine kinase phosphorylation motif	Periplasma, involved glucose metablism
PTS system lactose-specific EIICB component (STAAU)	P11162	K.EY*QLIILAPQVAS NY*EDIKQ#DTDR.L	Y515, Y528	2913.4	3.26	5.0E-15	Casein kinase II phosphorylation motif	Multi-pass membrane protein; involved in lactose transport

Y* = nitrotyrosine.

N^# = deamidated Asn.

Q^# = Deamidated Gln

Protein domain versus nitrotyrosine site

Protein-domain analysis demonstrated that each nitrotyrosine site was located within a specific protein domain and motif (Figure 1 and Table 1). Two of the identified nitrotyrosine sites also appear to be the targets of tyrosine phosphorylation, including lysozyme (nY₈₅ was located at the tyrosine kinase phosphorylation motif K-ywc-D-sg-Y₈₅; Figure 1F) and aldose 1-epimerase (nY₁₁₈ was located at the tyrosine kinase phosphorylation motif K-fsl-D-gkt-Y₁₁₈; Figure 1G). Nitrotyrosine (nY₇₉) was also detected in the tyrosine kinase phosphorylation motif (K-y₇₉wc-D-sg-Y) of lysozyme (Figure 1F). Nitrotyrosines (nY₅₁₅ and nY₅₂₈) appear in the PTS EIIB type-3 domain of PTS system lactose-specific EIICB component (Figure 1H), and nY₅₂₈ occurred in the casein kinase II phosphorylation motif (S-ny-E). The other identified nitrotyrosine sites were detected in the cytoplasmic region (nY₃₃) of multi-pass membrane protein progestin and adipoQ receptor family member III (Figure 1A), the KRAB domain (nY₄₁) of zinc finger protein 432 (Figure 1B), the functional chain (nY₂₂₉) of proteasome alpha 2 (Figure 1C), the mitochondrial complex I functional chain (nY₃₆ and nY₄₀) of NADH-ubiquinone oxidoreductase B14 (Figure 1D), and the EGF-like domain 3 (nY₁₀₃₅) of slit homolog 1 protein (Figure 1E). The relationship analyses of each nitrotyrosine site and protein domain suggest that each nitrotyrosine site resides within an important domain and motif of each protein; that correlation suggests the physiological significance of tyrosine nitration in each individual protein functional alteration.

Figure 1.

Nitrotyrosine was located within specific protein domains and motifs. Two nitrotyrosine sites (nY₇₉ and nY₈₅) were present in two individual peptides from lysozyme, respectively.

Metabolic networks that involve nitroproteins

Metabolic network systems that involve nitroproteins could reveal the significance of tyrosine nitration in human ex-smoker lung physiology. Pathway analysis suggested that those nitroproteins could be involved in multiple functional metabolic systems, including transcriptional regulation, mitochondrial complex, immune system, energy metabolism, protein metabolism, etc. (Table 1). Proteasome subunit alpha type 2 (PSMA2), which is nitrated, and is an important component of the proteasome complex, was identified (Figure 2A); the proteasome complex is involved in protein synthesis, cancer formation, and lung fibrosis disease; and the ubiquitin-proteasome pathway is involved in the mechanisms of reduced contractile protein content in the diaphragm of patients with mild to moderate COPD [20]. NADH-ubiquinone oxidoreductase B14, which is nitrated, is involved in the mitochondrial membrane respiratory chain NADH dehydrogenase system (Figure 2B), and is a component of the mitochondrial complex I that is involved in the apoptosis signaling pathways in lung diseases [21]; recent studies found that a cigarette smoke-induced blockade of the mitochondrial respiratory chain was involved in the mechanisms of increased lung cell apoptosis and necrosis in COPD patients [22]. Therefore, the nitration of that component of the mitochondrial respiratory chain could involve this process.

Figure 2.

Multiple metabolic networks that were involved in nitroproteins. A. Nitrated proteasome alpha 2-involved network. B. Nitrated NADH-ubiquinone oxidoreductase B14-involved network.

DISCUSSION

Oxidative/nitrative stress plays important roles in cigarette smoke-related inflammatory airway diseases, including COPD [3], such as an amplification of inflammation via the activation of transcription factors such as NF-kB and AP-1, reduction of antiproteases to accelerate the breakdown of elastin, increase of apoptosis and death in endothelial and epithelial cells, and even increased resistance of anti-inflammation processes to corticosteroids. Nitrotyrosine, as a biomarker of oxidative/nitrative stress, significantly increases in smoke-related inflammatory lung diseases [5]. The goal of this present study was to identify the targets of nitrotyrosine in human ex-smoker BALF to further clarify the mechanism of cigarette smoke-related inflammatory airway disease and to provide an optimized experimental approach and control reference for a COPD nitroprotein study.

Nitrotyrosine immunoaffinity enrichment was used to preferentially isolate nitroproteins from human ex-smoker BALF, and to improve the sensitivity to detect nitrotyrosine sites. The accumulated (n = 50) MS² spectrum of each tryptic peptide from the IP product was acquired with a vMALDI-LTQ to optimize the S/N and to improve the quality of each MS² spectrum to accurately identify nitrotyrosine sites. To select for low-abundance nitrotyrosine-containing peptides, Bioworks TuboSequest database searches, in combination with a manual inspection and interpretation of each MS/MS spectrum, identified nine nitropeptides (Table 1), with support of reliable amino acid sequence information (Supplemental Figures A–H). BLAST homolog analysis provided the statistically significant identification of nitrotyrosine sites in seven human BLAF proteins, with an NCBI BLAST E-value range from 5E-15 to 9E-21 (Table 1), except for proteasome subunit alpha type 2 (E-value = 4.9), which, however, was supported by the reliable amino acid sequence (Supplemental Figure C).

Bioinformatics analyses of protein domains and metabolic networks clarify the biological significance of identified endogenous BALF nitroproteins. Each nitrotyrosine site was located in an important functional and structural domain or motif of each protein (Figure 1), and nitroproteins are involved in multiple metabolic networks (Figure 2).

The biological significance of each identified nitrotyrosine-containing protein was rationalized into pathophysiological processes of cigarette smoke-related inflammatory pulmonary disease such as COPD (Figure 3). Cigarette smoke contains a variety of oxidants and nitrosants [1] that activates the alveolar macrophage, neutrophil, CD8⁺ lymphocyte, epithelial cells, and other inflammatory cells to further produce endogenous oxidants such as O₂^.−, ·OH, H₂O₂, and nitrosants such as NO·. Peroxynitrite (ONOO⁻), which is derived from the rapid reaction between NO· and O₂^.−, is a toxic oxidant and nitrosant, and is a primary source to nitrate a protein tyrosine residue. Our results showed that this nitration often occurred at specific protein domains or important motifs that could alter the protein functions (Figure 1) and could affect the corresponding functional networks. The nitrated proteasome subunit alpha type 2 (PSMA2) is involved in the ATP-dependent ubiquitin-proteasome pathway, which is an important intracellular nonlysosomal proteolytic pathway [23, 24], and which is also experimentally found to be involved in the processes of reduced contractile protein content in the diaphragm of COPD patients [20]. The nitrated NADH-ubiquinone oxidoreductase B14 is a component of the mitochondrial complex I and of the mitochondrial membrane respiratory chain NADH dehydrogenase system, which is involved in the mechanisms of increased lung cell apoptosis and necrosis in COPD patients [22]. The nitrated aldose-1-epimerase (mutarotase) is involved in the interconversion of alpha- and beta-hexoses, which are essential for normal carbohydrate metabolism and the production of complex oligosaccharides, and could affect energy metabolism and synthesis of cell-surface glycoproteins and glycolipids [25]. The nitrated PTS system lactose-specific EIICB component catalyzes the intake and phosphorylation of lactose, which is involved in sugar and energy metabolism [26, 27]. Lysozyme is induced by bacterial infection, is expressed in salivary glands and tubules, and has primarily a bacteriolytic function [28]; in tissues and body fluids, lysozyme is associated with the moncyte-macrophage system and enhances the activity of immunoagents, and contributes to the antimicrobial defense of the alveolar lining layer [29] and respiratory system defense [30–32]. Tyrosine nitration (nY₇₉ and nY₈₅), which occurred within the tyrosine kinase phosphorylation motif (K-y₇₉wc-D-sg-Y₈₅) of lysozyme (Figure 1F), could interfere with its bacteriolytic function and inhibit the activity of immunoagents, which could promote bronchoalveolar infection and inflammation. Slit homolog 1 protein, which was nitrated, is secreted and functions as a ligand for the Roundabout (Robo) receptor-signaling pathway, which is implicated in neurogenesis, angiogenesis, and the immune response [33]. For zinc finger 432, which is involved in the transcription regulation of a broad range of genes, nitration (nY₄₁) occurred within the kruppel-associated box (KRAB) domain (Figure 1B) that functions as a transcriptional suppressor [34, 35]; this nitration might impair transcriptional suppression of proinflammatory genes. Progestin and adipoQ receptor III (PAQR3) is a multiple-pass membrane protein [36], and interacts with ligand progesterone. Nitration (nY₃₃) occurred within the extra-membrane region (Figure 1A), and could interfere with receptor-ligand binding and the signal transduction of progesterone. Hormone-replacement therapy, such as the use of estrogen and progesterone, has been used in chronic airway diseases with protean effects [37–39]. Therefore, the nitrated PAQR could interfere with effects of hormone replacement therapy (such as progesterone) in chronic airway inflammatory diseases.

Figure 3.

Biological significance of tyrosine nitration in cigarette smoke-related inflammatory pulmonary disease.

In summary, this present study is the first report to discover the presence of, and the potential biological significance of, nitroproteins in human ex-smoker BALF. Nitrotyrosine sites occurred within specific protein domains and motifs that could alter protein functions, and that are involved in multiple metabolic networks, including transcriptional regulation, mitochondrial complexes, immune system, and energy metabolism. This present study clarifies contribution of oxidative and nitrative stress to cigarette smoke-related airway inflammatory diseases. Further investigation is required to determine the biological consequences of these tyrosine nitration events and the relevance to the pathophysiological mechanisms of chronic airway inflammatory diseases.

Supplemental Figures

The MS/MS spectra of nitropeptides

A representative MS/MS spectrum (Supplemental Figure A) and the data interpretation used to identify the nitrotyrosine - containing 28 amino acid peptide from progestin and adipoQ receptor family member III is described here in detail. A singly charged ion ([M + H]⁺, m/z = 3340.6) in the MS spectrum was selected to accumulate MS/MS spectra (n = 100) with a normalized collision energy = 35 arbitrary units, followed by a Bioworks TuboSequest analysis in Swiss-Prot Database. The singly charged b - and y - product ions (y9, y11, y12, y14, y15, y16, y18, y19, y21, y22, y26, y26, y26, b9, b12, b18, b21, b23, b25, b26, and b27) are labeled in the MS/MS spectrum with the corresponding amino acid sequence LYTY*EQIPGSLKDNPYITDGYRAYLPSR shown, where Y* = nitrotyrosine. Furthermore, the singly charged a - productions (a7, a9, a11, a12, a13, a14, a16, a19, and a24) were detected in the MS/MS spectrum. Three ion - series of singly charged b -, y -, and a -ions determined, with a high level of confidence, this nitropeptide amino acid sequence and the assignment of the nitrotyrosine site. This peptide sequence matched exactly to residues 30 – 57 of the protein, and tyrosine nitration was assigned to Y 33 (Table 1). Similar approaches were used to interpret the eight other MS/MS spectra from human ex - smoker BALF shown in Supplemental Figures B–H.

1. Progestin and adipoQ receptor family member III

2. Zinc finger protein 432

3. Proteasome alpha 2

4. NADH-ubiquinone oxidoreductase B14

5. Slit homolog 1 protein

6. Lysozyme

7. Aldose 1- epimerase

8. PTS system lactose - specific EIICB component