2 RESULTS AND DISCUSSION
2.6 Proteome analysis of nitration and related oxidatively modifications in cystic
2.6.5 Mass spectrometric identification of oxidatively modified proteins after affinity-
In addition to the possible cystic fibrosis protein nitration characterization by 2-D gel and Western blot, the affinity-SDS PAGE was employed in this work from two reasons: (i), because the level of oxidative modification is very low and the sample amount can be increased by affinity enrichment, (ii), many of the oxidatively modified proteins present in the sputum could/may have a pI that exceed the range in the 2-D gel separation. Approximately 60 µg proteins extracted from sputum were precipitated in cold acetone for 6 hrs at 4oC and resolubilized in the PBS buffer.
Soluble fractions were loaded onto the affinity column immobilized anti-3-nitro tyrosine antibody and incubated for 2 hrs at 25oC. After the first incubation the column was washed with approximately 100 ml PBS buffer pH 7.4 followed by a second inculation of protein solution. The unbound fractions were washed away,
while the bound proteins were eluted with 250 µl 0.1% TFA performed two times. The column was then washed again with PBS buffer and stored at 4oC for further use. A schematic representation of affinity-SDS PAGE experiment workflow is shown in Figure 2.57.
Washing Elution with 0.1% TFA
Y
Washing Elution with 0.1% TFA
Binding sputum
Washing Elution with 0.1% TFA
Y
Washing Elution with 0.1% TFA
Binding sputum
Figure 2.57. Experimental workflow of an affinity-SDS PAGE experiment using cystic fibrosis sputum protein mixture. Approximately 60 µg of protein mixture was loaded on NHS-immobilized sepharose anti-3-nitrotyrosine antibody (MAB5404) and incubated for 2 hrs. The supernatant, wash and elution fractions were separated by SDS-PAGE and proteins were identified by LC-tandem mass spectrometry
The supernatant, wash and elution fractions were lyophilized, resolubilized in running buffer and loaded on top of 12% polyacrylamide gel. As can be observed in Figure 2.58 the wash fractions are clean (lane 2); elution fractions (lane 3) contain two intense bands and several other weak bands.
Figure 2.58. SDS-PAGE separation of supernatant (A), wash (B) and elution (C) fractions collected after affinity experiment; gel stained with Coomassie blue (left) and Western blot experiment using anti-3-nitrotyrosine antibody (MAB5404) (right). The most intense bands (1 and 2) were cut out, destained, in-gel digested with trypsin and analysed by nano-LC-tandem mass spectrometry
Mass spectrometric analysis of band 1 revealed the identification of lactotransferrin. The sequence coverage obtained by trypsin digestion was 46% from full-length sequence and it is shown in Figure 2.59. Lactotransferrin is a globular glycoprotein with a molecular mass of 80 kDa protein having a calculated pI of 8.5.
Lactotransferrin is a component of immune system of human body having an antimicrobial activity and it is part of the innate defense, mainly at mucoses.
Antimicrobial activity of lactotransferrin comes from its capabilities of iron-binding, which is a necessary chemical element for bacterial growth. Lactotrnsferrin bearing oxidized iron binds to lipopolysaccharidic bacterial walls affecting membrane permeability and eventually cell breackdown [227]. It contains 20 tyrosine residues and 18 of them were encompassed in the matched peptides. A number of three tyrosines (Tyr-343, Tyr-686 and Tyr-417) were identified to be oxidized to hydroxytyrosine. The mass spectrometric identification of tyrosine oxidation is shown in Figure 2.59 and 60. The hydroxy-tyrosine modification structures were assigned by manual analysis of the fragment ions resulted after CID fragmentation of the peptide precursor ion containing modified tyrosines versus unmodified peptides.
1 MKLVFLVLLF LGALGLCLAG RRRSVQWCAV SQPEATKCFQ WQRNMRKVRG 51 PPVSCIKRDS PIQCIQAIAE NRADAVTLDG GFI EAGLAP KLRPVAAEV
GTERQP TH AVAVVKKG GSFQLNELQG LKSC TAGWNVPIGT LRPFLNWTGP PEPIEAAVAR
SSQEPYFS SGAFKCLRD GAGDVAFIRE STVFEDLSDE AERDE ELLC SVN GKEDAIWNLL RQA
FQLFGS PSGQKDLLFK DSAIGFSRVP IDSGL LG SG FTAIQNL RKSEEEVAAR RARVVW A G EQELRKCNQW SGLSEG I LV KGEADA MSLDGG V T AGKCGLVPVL AEN KS P VEG LAVAVV RRSDTSLTWN SVKGK
KFDE Q SCAPGSDPRS NLCALCIGDE QGE G TGA FRCLAENAGD VAFVKDVTVL QNTDGNNNEA WAKDLKLA K PVTEARSCH MDKVERLKQ VLLHQQAKF
K LGPQ VAGI TNLKK
Y Y
101 Y R YY HTGLRR
151 FFSASCVPGA DKGQFPNLCR LCAGTGENKC
201 AF Y Y
251 PDNTRKPVDK FKDCHLARVP SHAVVAR QEKFGKD
301 KSPK PR Y Y
351 C V SVTC SSASTTEDC
401 A L Y Y Y QQSS DPDPNCVDR
451 Y KSCHT AVDRTAGWNI PMGLLFNQTG
501 SC YFS NKCVPNS NERYY Y
551 DF ALLCLDGKR
601 L AMAPNHAVVS R G RNGSDCPDKF
651 CLFQSETKNL LFNDNTECLA RLHGKTTYE Y Y CSTSP 701 LLEACEFLRK
Figure 2.59. Mass spectrometric identification of lactotransferrin from band 1. After excision from 1-D gel, the protein spot was digested with trypsin, and measured by LC-tandem mass spectrometry. In the amino acids sequence of the protein, the identifiedpeptides are shown in red. The CID fragmentation mass spectrum of ion 1052.541 (2+) enable the unambiguous identification of hydroxytyrosine (A), by comparison with the unmodified peptide (B).
Figure 2.60. LC-tandem mass spectrometry analysis of tryptic peptides revealed the formation of hydroxytyrosine at Tyr-686 and Tyr-417 proved by the CID fragmentation of double charge precursor ion 776.923 (2+) (top) and 918.902 (2+) respectively (down).
The tryptic peptide (IDSGLYLGSGYFTAIQNLR) (333-351) containing two tyrosine amino acid residues was observed at m/z 1052.541 (2+) and the complete series of b and y ions shows unambiguously that only Tyr-343 is modified. A further example is the modification of Tyr-686 identified by the CID fragmentation of tryptic peptide (YLGPQIVAGITNLK) at m/z 776.923 (2+). In addition, the mass spectrometric analysis of the tryptic peptide (406-423) revealed the identification of hydroxy-tyrosine occurring at Tyr-417. In this case the CID fragmentation of peptide precursor ion at m/z 918.902 (2+) showed incomplete series of b and y ions, but enough to assign the modification site. The y8 fragment ion, which shows the modification at Tyr-417 is missing, whereas the presence of b11-b14 ions enables the
assignment of modification site. In addition, Met-411 was identified to be oxidized to Met sulfoxide.
The SDS-PAGE characterization of affinity experiment (elution fractions) shows also a thick band around 25 kDa. The corresponding band was also cut out, destained and digested with trypsin. The LC-tandem MS analysis of tryptic digest mixture has led to the identification of three proteins (azurocidin, myeloblastin and cathepsin G). In addition to this a number of weak bands could be observed in the elution fraction (Figure 2.58 lane C), however, no MS identifications was possible, therefore, a further series of affinity-mass spectrometry were employed. Sputum protein mixture were solubilized in PBS buffer and incubated on the anti-3-nitrotyrosine affinity column in the same manner as described before. The elution fractions were digested with trypsin followed by direct analysis using nano-LC-tandem MS. Besides the proteins identified in bands 1 and 2, LC-nano-LC-tandem MS analysis of revealed the identification of other nitrated protein candidates which are summarized in Table 2.11.
Table 2.11. Mass spectrometric identification of oxidative modified proteins after affinity enrichment using anti-3-nitro tyrosine antibody Human myeloperoxidase isoform C 9.4 53 70 65 gi7766942
Human lactotransferrin 8.5 78 55 52 gi75766355
Chain A H253 N terminal labe of
lactotransferrin 9.2 37 29 52 gi15781384
Cationic antimicrobial protein CAP
The Western blot of an affinity experiment shows in the elution fractions a number of bands, which corresponds to the molecular masses of the proteins shown in the Table 2.11. All relevant spectra of tyrosine containing peptides are covered in protein identified peptides and they were verifired manually, however, no nitration site could be assigned mainly because the protein mixture is still very complex even after affinity purifications step. In any case the affinity experiments of cystic fibrosis sputum protein enable identification of presumable nitrated protein candidate, which represents an important step aiming the identification of protein nitrations. The
sample complexity represents an important obstacle in the direct identification of nitration and purification of each of the nitrated protein mentioned above followed by affinity enrichment of peptides is a crucial prerequisite [228, 229].
3 3.1
EXPERIMENTAL PART Materials and reagents
IPG strips (3 – 10) were purchased from Bio-Rad (München, Germany), Millipore Pipette Tips C18 were from Millipore, carrier ampholytes (Servalyt 3 – 10) were from Serva (Heidelberg, Germany). Trizma®base, TEMED, Coomassie Brilliant Blue G and R, phosphoric acid 85% (H3PO4), ACN, TFA were from Sigma (St. Louis, MO, USA). The 30% acrylamide/0.78% N, N’-methylenebisacrylamide solution (37.5:1), DTT and CHAPS were purchased from Genaxxon Biosciences (Biberach, Germany). Ammonium sulfate - (NH4)2SO4, glycerol, glycine, SDS, TCA and EDTA were from Roth (Karlsruhe, Germany). Iodoacetamide (IAA), methanol (CH3OH), ammonium hydrogen carbonate (NH4HCO3), agarose and bromphenol blue were from Fluka (Buchs, Switzerland). Trypsin was purchased from Promega (Madison, WI, USA). Urea and thiourea were from Merck (Darmstadt, Germany). OxyBlotTM Protein Oxidation Detection Kit from Chemicon (Temecula, CA/USA) 2,5-dihydroxybenzoic acid (DHB) was from Bruker Daltonics (Bremen, Germany).
Millipore water (Millipore, Bedford, MA, USA) with a minimum electric resistance at 18.2 MΏ (Ώ: Ohm) was used for all solutions.
3.2 Sample preparation for proteome analysis
3.2.1 Muscle sample preparation using denaturing conditions
Samples were provided by the Department of Animal Breeding, University of Bonn, Germany taken from Longissimus dorsi muscle at the position of last rib collected 45 min., 12, 24 and 48 hours post-mortem. The snap-frozen pig muscle was weighted (average 150 mg) and kept at -80oC until further work-up. Muscle sample was solubilized in 8 M Urea, 2 M Thiourea, 70 mM DTT, 2 % CHAPS and 0.5
% Servalyte (3-10), protease inhibitor cocktail (complete); Weight-dependent (in mg) buffer volumes (in µl) were: lysis buffer: 9 x sample weight; complete: 0.04 x sample weight. Homogenization was performed in a hand-held glass homogenizer. After homogenization the samples were centrifuged at 4oC for 20 min at 15000 x g to remove unextracted cellular components and insoluble high-molecular weight proteins and protein complexes. The supernatant was removed and stored at -80oC until use.
3.2.2 Muscle sample preparation using non-denaturing conditions
Samples were taken from Longissimus dorsi muscle between the 13th and 14th rib and pH, meat conductivity and meat colour groups were measured using Star-series equipment (Rudolf Matthaeus Company, Germany). The muscle pH values measured 45 min., 12 and 24 hours post-mortem. Drip loss was scored using a bag method by a size-standardised sample from Longissimus dorsi that was collected 24 hours post-mortem. The samples were weighted, suspended in a plastic bag, held at 4oC for 48 hours and re-weighted at the end of holding time. Drip loss was calculated as a percentage of weight loss based on the start weight of the sample. In both (high and low drip loss) samples for homogenization was used the same lysis buffer. The snap-frozen pig muscle was weighted (average 150 mg) and kept at -80oC until further work-up. Briefly, muscle sample was homogenized in: 50 mM Tris-HCl, pH = 7.6, 150 mM NaCl, 1 % (w/v) CHAPS, 1 % (v/v) Triton X-100, 5 mM NaF, 2 mM activated Na3VO4 containing Complete Protease Inhibitor Cocktail (Roche). Weight-dependent (in mg) buffer volumes (in µl) were: lysis buffer: 9 x sample weight; complete: 0.04 x sample weight. After homogenization the samples were centrifuged at 4oC for 20 min. at 15000 x g to remove unextracted cellular
components, insoluble high-molecular weight proteins and protein complexes, and were then separated by 2-DE. The supernatant was removed and stored at -80oC until use.
3.2.3 Sample preparation of sputum from Cystic Fibrosis patients
The sputum sample collected from a 28 years old CF male patient chronically infected with P. Aeruginosa and it was provided from the Institute of Medical Microbiology and Hygiene University of Tübingen. The firsts proteomics studies were focused mainly on optimizations of both homogenization and solubilization of sputum proteins. In this respect two different lysis buffers were tested: lysis buffer 1 (DTT 1mg/ml) and lysis buffer 2 (DTT 1mg/ml + 100 µg/ml DNase). The protein concentration found was 4.2 mg/ml using lysis buffer 1 and 3.5 mg/ml in lysis buffer 2. The use of DNase in the lysis buffer provided a good separation of protein from DNA and also improved protein solubility.
3.3 Labelling methods of protein carbonyl groups for immunological detection
3.3.1 Pre-isoelectric focusing labelling of protein carbonyl using 2,4-dinitrophenyl hydrazine
2,4-dinitrophenylhydrazine (DNPH) was prepared as a 10 mM solution in 10% HCl. Five hundred micrograms of muscle proteins were dissolved in 12% SDS mixed with an equal volume of DNPH and incubated at room temperature for 20 min.
Samples used for 1-DE were loaded directly on SDS-PAGE gels. Proteins used for IEF were precipitated by addition of ice cold acetone. Samples were incubated on ice for 20 min., and precipitates were centrifuged at 15 000 x g for 5 min. The precipitated proteins were washed with 1 mL of 1:1 v/v ethanol/ethyl acetate followed by centrifugation for 5 min at 15 000 x g three times. Precipitated samples were resuspended in 2-DE sample buffer and subjected to electrophoresis. Peptide derivatives produced by reaction with DNPH were immunodetected by an antibody specific to the DNP moiety attached to proteins using a commercial kit (Chemicon, Temecula, CA).
3.3.2 Post-isoelectric focusing labelling of protein carbonyl using 2,4- dinitrophenyl hydrazine
To identify carbonyl groups introduced into the amino acids side chain after oxidative modification of proteins, 2-D oxyblot analysis was performed, as previously described [22]. Peptide derivatives produced by reaction with DNPH were immunodetected by an antibody specific to the DNP moiety attached to proteins using a commercial kit (Chemicon, Temecula, CA). Briefly, approximately 500 µg of muscle proteins solution was loaded on linear (3-10) IPG strips and isoelectrically focused. The IPG strips were then placed in 15 ml test tubes and incubated for 20 min. in 2 N HCl with 10 mM DNPH at 25oC. After the reaction, the samples were washed for 15 min with 2 M Tris-base/30 % glycerol. The IPG strips were then prepared for molecular weight separation. For estimation of specificity, the following oxidized proteins were included as positive controls: Phosphorylase B, 97.4 kDa;
bovine serum albumin, 68 kDa; ovalbumin, 43 kDa; carbonic anhydrase 29.1 kDa;
trypsin inhibitor, 21 kDa. For protein identification, spots were excised from the 2-D gels obtained with non-DNPH-treated samples and analyzed by mass spectrometry.
3.4 Chromatographic and electrophoretic methods for protein separation and staining procedure
3.4.1 Reverse Phase High Performance Liquid Chromatography
High-performance liquid chromatography (HPLC) was used in the present work to separate components of a mixture by using different chemical interactions between the protein/peptides of interest and the chromatography column. Molecules bearing some extent of hydrophobic character, such as proteins and peptides, can be separated by reverse phase chromatography with good resolution and recovery. RP-HPLC is based on the principle of hydrophobic interactions, resulting from repulsive forces between a polar eluent and the non-polar stationary phase.
Usually, the sample is injected into the HPLC column directly into the stream of mobile phase and it is retarded by specific chemical or physical interactions with the stationary phase as it pass through the length of the column. There are mainly three types of columns namely, C4, C8, and C18 that interact with the hydrophobic moieties of the analyte. The C4 and C8 are used for protein separation, while C18 is used for small peptides. Peptides and/or proteins are eluted from the reverse phase column with aqueous solvents containing an ionic modifier to adjust the pH and an organic modifier to displace and elute the peptide by increasing gradient in the organic modifier as shown in Table 3.12.
Table 3.12. The gradient used for the separation of synthetic peptides Time %A %B
0 100 0
5 100 0
85 20 80
90 0 100
95 100 0
100 100 0
Prior using aqueous solutions, they were well degased by vacuum combined with sonication. The sample was dissolved in 0.1% TFA compatible with the mobile phase (usually the components of the gradient starting point) to avoid precipitation in the pores of the column packing. All RP-HPLC experiments were performed on a Bio-Rad system (Bio-Rad Laboratories, Richmond, CA) using PLRP-S column (250 x 4.6mm, 300 Å, 5µm) contain rigid macroporous spherical particles of polystyrene/divinylbenzene (Polymer Laboratories, Darmstadt, Germany). The
samples were dissolved in eluent A and the peaks were detected at two different wavelengths λ = 365 nm for nitrated peptides and λ = 220 nm for non-nitrated peptides. The purification of polypeptides was carried out on a Knauer system (Bad Homburg, Germany) using a preparative C18 column (GROM-SIL 120 ODS-4 HE, 10 µm, 250 x 20 mm, pore size 120 Å; Herrenberg-Kayh, Germany) The same eluents as described above with appropriate linear gradients were applied. Flow rate was 10 mL/min for preparative HPLC. Peaks were detected at 365 nm and 220 nm.
3.4.2 Sodium dodecyl sulphate – polyacrylamide gel
A very common method for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. The method is called sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS (also called lauryl sulfate) is an anionic detergent, meaning that, when dissolved, its molecules have a net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in proportion to its relative molecuar mass. The negative charges on SDS destroy most of the complex structure of proteins, and they are strongly attracted toward an anode (positively-charged electrode) in an electric field.
SDS PAGE was performed accoding to Laemmli using the Mini-Protean II gel electrophoresis system (Biorad, München, Germany) by pouring SDS-polyacrylamide gels between two glasses plates resulting a gel were 90 x 60 x 1 mm in size. The stacking gel was poured over the top of the separating gel, which allows the proteins in a lane to be concentrated into a tight band before entering the separating gel. The acrylamide concentration stacking gel is lower than the concentration of separation gel and thus a larger pore size, lower pH and different ionic content. The lower the concentration of acrylamide the larger size of the pores in the gel is created, therefore gels with a low percentage of acrylamide are typically used to separate large proteins, while high percentage gels were used for small proteins. Table 3.13 shows the different acrylamide concentrations used in the present work.
Table 3.13. Composition of SDS-polyacrylamide gels according to Laemmli.
Stacking gel Separating gel Monomer concentration
c 30% (w/v) Acrylamide, 0.8% (w/v) N, N’- Methylenebisacrylamide
d 10% (w/v) Ammonium persulfate
e N, N, N’, N’- tetramethylethylenediamine
Muscle proteins were solubilised in a SDS reducing buffer (50 mM Tris-HCl, 4 % (w/v) SDS, 25 % (w/v) glycerol, 6M urea, 0.02 % (w/v) bromophenol blue, pH 6.8). The running buffer used contained of 25 mM Tris, 192 mM Glycin and 0.1 % SDS. Gel electrophoresis was carried out using a Power/PAC 1000 power supply (Bio-Rad, München, Germany) at a constant voltage of 60 V for approximately 30 min, until the tracking dye entered the separating gel, and at 110 V for ca. 2 hrs., until the tracking dye reached the anodic end of the separating gel. After separation, proteins were visualised by sensitive colloidal Coomassie or transferred on PVDF membrane for Western blot experiments. The Coomassie gels were scanned using a GS-800 Calibrated Imaging Densitometer (Bio-Rad, München, Germany). The molecular weights of proteins of interest were estimated by running standard proteins of known molecular weights, summarized in Table 3.14.
Table 3.14. Protein molecular weight marker for SDS-PAGE analysis (PS-101, Jena Bioscience) Molecular weight marker proteins Molecular weight (Da)
Phosphorylase b 97,400
Bovine serum albumin 66,200 Alcohol dehydrogenase 37,600
Carbonic anhydrase 28,500
Myoglobin 18,400 Lysozyme 14,400
3.4.3 Two- dimensional gel electrophoresis
Two-dimensional gel electrophoresis (2-D gel electrophoresis) is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. By this method proteins are separated in two steps, according to two independent properties: the first-dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pI); the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (Mw). In this way, complex mixtures consisting of thousands of different proteins can be resolved and the relative amount of each protein can be determined.
The procedure involves placing the sample in gel with a pH gradient, and applying a potential difference across it. In the electrical field, the protein migrates along the pH gradient, until it carries no overall charge. This location of the protein in the gel constitutes the apparent pI of the protein. The IEF is the most critical step of the 2-D electrophoresis process. The proteins must be solubilized without charged detergents, usually in high concentrated urea solution, reducing agents and chaotrophs. To obtain high quality data, it is essential to achieve low ionic strength conditions, before the IEF itself. Since different types of samples differ in their ion content, it is necessary to adjust the IEF buffer and the electrical profile to each type of sample. The main experimental steps involved in 2-D gel electrophoresis are shown in Figure 3.61.
Figure 3.61. Two-dimensional gel electrophoresis experimental steps. Crude protein mixture extracts are separated in the first dimension according to their specific isoelectric point (pI), followed by the separation in the second dimension by their molecular weight (Mw).
IEF was carried out using a Multiphor horizontal electrophoresis system (Amersham Biosciences, Uppsala, Sweden) using 17 cm IPG strips (pH range 3-10) with the sample being applied overnight using the in-gel rehydration method. The reswelling solution contained 7 M urea, 2 M thiourea, 4 % CHAPS, 0.3 % DTT, 2 % Servalyt (3-10) and a trace of bromophenol blue. Rehydrated strips were run in the first dimension for about 30 kVh at 19 oC. After focusing, the IPG strips were equilibrated for 30 min in 6 M urea, 30 % glycerol, 2 % (w/v) SDS, 0.05 M Tris-HCl (pH 8.8), 1 % (w/v) DTT and a trace of bromophenol blue, and they were then run for further 30 min in the same solution except that DTT was replaced by 4.5 % (w/v)
IEF was carried out using a Multiphor horizontal electrophoresis system (Amersham Biosciences, Uppsala, Sweden) using 17 cm IPG strips (pH range 3-10) with the sample being applied overnight using the in-gel rehydration method. The reswelling solution contained 7 M urea, 2 M thiourea, 4 % CHAPS, 0.3 % DTT, 2 % Servalyt (3-10) and a trace of bromophenol blue. Rehydrated strips were run in the first dimension for about 30 kVh at 19 oC. After focusing, the IPG strips were equilibrated for 30 min in 6 M urea, 30 % glycerol, 2 % (w/v) SDS, 0.05 M Tris-HCl (pH 8.8), 1 % (w/v) DTT and a trace of bromophenol blue, and they were then run for further 30 min in the same solution except that DTT was replaced by 4.5 % (w/v)