Detection and identification of oxidatively modified proteins using anti-3-nitro

The major goals of this proteomics study were to provide a 2-D proteome map of the cystic fibrosis sputum sample in order to detect the presumable nitrated proteins using specific anti-3-nitro tyrosine antibodies and to identify them by mass spectrometry. For this purpose, sputum proteins were extracted from a 28 years old cystic fibrosis patient chronically infected with P. aeruginosa and separated by 2-DE.

Proteins are the ultimate product of gene expression and the development of prognostic tests and drugs for cystic fibrosis will occur through a greater understanding of proteins and their interactions within the lung environment.

Proteomics provides the ability to characterize proteins and their post-translational modifications and offers a greater understanding of the physiology of the lung environment. For complex solutions, such as sputum, 2-D gel electrophoresis represents a key technology of choice for arraying and characterizing constituent proteins, and has been used to help characterize protein expression in bronchoalveolar lavage fluid from individuals with cystic fibrosis [37, 210] . This study, which represents the first proteomic study to address differential sputum proteome in the context of subjects with cystic fibrosis versus control individuals, aims to identify protein biomarkers that are indicative of an acute pulmonary exacerbation.

Sputum proteins were suspended in lysis buffer consisting of DTT (1 mg/ml) and DNase (100µg/ml). The suspension was incubated for 30 min. at 37^oC and centrifuged for 10 min. at 5000 rpm. to sediment unsolubilized material. The protein content in the supernatant was determined by the Bradford method [111]. After concentration assay proteins were precipitated using cold acetone for 6 hrs.

Approximately 400 µg total protein mixture was applied to IPG strips (3-10) and proteins were separated in the 1^st dimension according to their specific pI. After equilibration, the IPG strips were applied on top of 12 % polyacrylamide gel and run for 5 hrs. A number of two gels were run in parallel; one of them was stained with Silver staining and the other was electroblotted on PVDF membrane for Western blot experiments with anti-3-nitro tyrosine antibody (MAB5404) Figure 2.51.

Figure 2.51. (A) 2-D PAGE gel separation of proteins stained with silver from cystic fibrosis patient sputum chronically infected with P. aeruginosa. The proteins are separated by isoelectric point in the first dimension using IPG strips (3-10) and by molecular weight in the second dimension, (B) Western blot experiment using anti-3-nitrotyrosine antibody for detection of nitrated proteins

Figure 2.51a displays the 2-D proteome map of cystic fibrosis sputum sample. Proteins are mostly concentrated in the pI range 4-8 and at the lower molecular weight. Mass spectrometric analysis of the most abundant proteins (spot 1 and 2) revealed the identification of two calgranulin isoforms; S100 A8 (Calgranulin A) and S100 A9 (Calgranulin B), which are specific proteins found in cystic fibrosis sputum patients chronically infected witk P. aeruginosa [211]. Mass spectrometric identification of calgranulin B from database is shown in Figure 2.52.

179.2 345.3 436.3 586.9 586.9

624.7 879.9

1544.2 1821.0

200 400 600 800 1000 1200 1400 1600 1800 m/z

145.

400 600 800 1000 1200 1400 1600 1800 m/z

145.

Figure 2.52. Identification of Calgranulin B by LC-MS/MS of the in-gel digest spot 2. Total ion chromatogram, database search result using Mascot software; a number of 5 tryptic peptides were matched covering 49% of the protein full-length sequence, precursor ion at m/z 586.6 (3+) isolated for subsequent CID fragmentation corresponding to the (58-85) peptide

Calgranulin B was identified by mass spectrometric anlysis of the resulting tryptic after in-gel digestion based on 5 peptides covering 49% of the protein full-length sequence. Calgranulins belong to a class of calcium-binding proteins known as the S100 protein family [212]. The biological function of these proteins is performed by binding calcium, resulting in the exposure of the hydrophobic domains, which interact with the hydrophobic domains of specific effector proteins regulating their activity. They are the major binding protein of neutrophiles and monocytes.

Calgranulin B in complex with its binding partner Calgranulin A (S100A8), promotes polymerization of microtubules [213]. Although, the two calgranulins isoforms are very abundant, they show no immuno-reactivity against anti-3-nitrotyrosine antibody, meaning that they are not nitrated Figure 2.51b. The non-nitrated proteins identified by MS are summarized in Table 2.9.

Table 2.9. Identification of sputum proteins which shown no immunoreactivity against anti-3-nitro

Patients with cystic fibrosis are highly susceptible to respiratory tract colonization and infection, decreased host defenses and overly exuberant inflammatory response that may result in large amounts of neutrophilic myeloperoxidase in airway secretions [214]. The resulting magnitude of the neutrophilic responses and their accompanying proteolytic and oxidative processes are believed to play a major role in the progressive destruction of lung tissues, which characterize cystic fibrosis [215]. As a result of intense proteolytic activity many proteins are truncated. For example, myeloperoxidase is a 83 kDa protein having a pI around 9.1 and it was identified at a molecular weight (in the gel) of approximately 12 kDa and a pI of 5.5 (spot 4). This proves the intense proteolytic activity that takes place in cystic fibrosis patient respiratory tract. Myeloperoxidase, a heme enzyme, is released by activated neutrophils in the extracellular milieu, where it uses H2O2 and chloride anion (Cl^-), forming hypochlorous acid (HOCl). HOCl is a powerful oxidant and reacts with amines to form chloramines [216]. The increase of myeloperoxidase leads to a decreased level of exhaled NO in cystic fibrosis patients. A further enzyme identified is superoxide dismutase [Mn] (mitochondrial) with the physiological fuction of keeping low the level of superoxide (O2• –) releasing hydrogen peroxide as a major decomposition product. LC-MS/MS provided the identification of superoxide dismutase located in the gel at a pI of 6.8 and Mw of 22 kDa.

Western blot experiments using anti-3-nitro tyrosine antibody enable detection of 6-7 proteins spots from which a number of 3 were successfully identified by mass spectrometry. All identified cystic fibrosis sputum nitrated proteins are summarised in the Table 2.10 together with their molecular weight, isoelectric point, sequence coverage, number of matched peptides and the protein accession number from SWISS-PROT database.

Table 2.10. Nano-tandem LC-MS/MS identification of protein bands from the elution fractions after affinity purification using anti-3-nitro tyrosine antibody (MAB5404)

Spot Protein Mw

(kDa) pI Sequence

coverage % Peptides

matched Accession no.

5 Hemoglobin subunit beta 16 6.7 97 40 P68871 10 Ig gamma 1 chain C region 36 8.4 64 49 P01857 12 Leukocyte elastase inhibitor 42 5.9 84 70 P30740

The identification of proteins showing immunoreactivity against anti-3-nitrotyrosine antibody (MAB5404) was conducted by nano-LC-tandem mass spectrometry, firstly, because the spot intensity of these proteins was very low and, secondly, nano-LC-tandem mass spectrometry represents a powerful tool for structural characterization of oxidative post-translational modifications as has been shown in the previous chapter.

Of particular interest among the identified oxidative modified proteins is leucocyte elastase inhibitor; a major serine protease inhibitor usually found in human plasma and in lower respiratory tract [217], which leads to the fragmentation of elastin, fibronectin and cell receptors on neutrophils [218]. The mass spectrometric identification leucocyte elastase inhibitor is shown in Figure 2.53. This protein contains 10 tyrosine residues, which were covered in the matched peptides. All the peptides containing tyrosines were verified manually in an attepmt to find possible nitration sites. A number of two tyrosines (Tyr-272 and Tyr-209) were found to be oxidized to hydroxy-tyrosine.

MEQLSSANTR FALDLFLALS ENNPAGNIFI SPFSISSAMA MVFLGTRGNT

AAQLSKTFHF GAS ILKLAN RL GEKT NF

LPEFLVSTQK T GADLASVD FQHASEDARK TINQWVKGQT EGKIPELLAS GMVDNMTKLV LVNAI FKGN WKDKFMKEAT TNAPFRLNKK DRKTVKMM KKKFA G IE DLKCRVLELP QGEELSMVI LLPDDIEDES TGLKKIEEQL TLEKLHEWTK PENLDFIEVN VSLPRFKLEE S TLNSDLAR LGVQDLFNSS KADLSGMSGA RDIFISKIVH KSFVEVNEEG TEAAAATAGI ATFCMLMPEE

NTVEEVHSRF QSLNADINKR Y Y Y Y

Y YQ

Y Y Y

Y NFTADHPFLF FIRHNSSGSI LFLGRFSSP 1

151 201 251 301 351 101 51

Figure 2.53. Mass spectrometric identification of leukocyte elestase from spot 12 showing the full-length protein sequence (top); the matched peptides are highlighted in red and tyrosine residues representing possible nitration sites in blue. After excision from 2-D gel, the protein spot was digested with trypsin. LC-tandem mass spectrometry analysis of tryptic (266-290) revealed the formation of hydroxytyrosine at Tyr-272 proved by the CID fragmentation of double charge precursor ion 901.452 (A) and, the Tyr-209 in the tryptic peptide (204-213) at m/z 617.801 (C). In both cases the unmodified peptides were identified (B and D).

As has been shown in previous studies mass spectrometric characterization of nitration sites is difficult as the nitration level is usually very low [219]. Hence, the the mass spectrometric identification of nitration is even more difficult as this modification may be overlapped by other oxidative modifications occurring at tyrosine residues such as hydroxytyrosine formation.

2.6.4 Affinity-mass spectrometric characterization of anti-3-nitro tyrosine