in Spinal Cord Injury of the Rat - American Chemical Society

Nitric Oxide and Oxygen Radical Attack on GDP-Dissociation Inhibitor 2 (GDI-2) in Spinal Cord Injury of the Rat Julius Paul Pradeep John,† Oliver Pintsov,‡ Alexander Petter-Puchner,‡ Heinz Redl,‡ Arnold Pollak,† Wei-Qiang Chen,† and Gert Lubec*,† Department of Pediatrics, Medical University of Vienna, Vienna, Austria, and Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria Received November 22, 2006

Protein oxidation and nitration have been described during spinal cord injury (SCI) in animal models. Herein, mass spectrometry unambiguously identified GDP-dissociation inhibitor-2 (GDI-2) in SCI with post-translational modifications of 3-aminotyrosine (8 h post-injury) and an acrolein adduct of GDI-2 (72 h post-injury). On the basis of mass spectrometry evidence, we conclude that lipid-peroxidation and protein nitration do take place on an important signalling protein that may be prevented by specific experimental therapeutic interventions. Keywords: Spinal cord injury • Up-regulation • de novo sequence • 3-aminotyrosine • Acrolein

Introduction Apart from quantitative changes of protein levels, qualitative alterations in terms of post-translational modifications (PTMs) have been reported in spinal cord injury (SCI). The reported PTMs associated with SCI were induced by active oxygen species or nitric oxide. PTMs reflecting protein oxidation included oxidative modifications of oxidation-sensitive amino acids tryptophan, tyrosine, phenylalanine, sulfur-containing amino acids cysteine, cystine, and methionine, lysine, proline, histidine, and arginine.1,2 In SCI, no specific oxidation-induced PTMs have been demonstrated and protein oxidation is determined by detection of protein carbonyl content,3,4 and only Luo and co-workers reported accumulation of acrolein-protein adducts by Western blotting and immunohistochemistry.5 Presently, no site-specific identification of oxidative modification was published to the best of our knowledge. As to nitric oxide-induced protein modifications (protein nitration), formation of 3-nitrotyrosine (3-NT) has been reported in SCI by Liu and co-workers using HPLC of a protein hydrolysate,6 and Bao and co-workers showed 3-NT in SCI of the rat by immunohistochemistry.7 No mass spectrometrical verification of PTMs in SCI has been demonstrated, and this formed a rationale to carry out the present study. Protein profiling in SCI of the rat revealed that, at 8 h following the traumatic lesion, levels of a signalling protein, GDP-dissociation inhibitor-2 protein (GDI-2) (synonym: Rab GDP dissociation inhibitor beta), were increased about 3-fold, and we therefore decided to use this potentially * Corresponding author: Prof. Gert Lubec, Dept. of Pediatrics, Medical University of Vienna, Waehringer Guertel 18, 1090 Vienna, Austria. Tel: +431-40400-3215. Fax: +43-1-40400-3200. E-mail: [email protected]. † Medical University of Vienna. ‡ Ludwig Boltzmann Institute for Experimental and Clinical Traumatology.

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important signalling structure to study the presence of oxidation- and nitration-induced PTMs in SCI. We observed two PTMs representative of protein oxidation and nitration, but failed to show the presence of the nitration product 3-NT using specific MALDI-TOF/TOF methodology. As we prepared twodimensional gels for the protein extracts of SCI samples under reducing conditions, however, we were finally able to detect a parameter for protein nitration, the 3-NT reduction product 3-aminotyrosine on tyrosine Y-224.8,9 Protein oxidation in SCI was represented by the lipid peroxidation-induced acroleinadduct of lysine (K-174) of rat-GDI-2. This report may be helpful as an analytical tool for the determination of oxidative and nitration changes of proteins in SCI and other diseases with involvement of active oxygen species and or nitric oxide attack.

Experimental Section Animal Experiments. Male Sprague Dawley (SD) rats weighing 200-250 g were obtained from the Division for Laboratory Animal Science and Genetics, Core Unit for Biomedical Research of the Medical University of Vienna (Himberg, Austria). Animals were matched according to baseline characteristics (age and weight). All reagents used were of analytical grade. The study protocol was approved by the City Government of Vienna (Department 58) and followed its regulations on animal trials. Surgical interventions, animal care, and euthanasia, as well as sample preservation, were conducted by investigators of the Ludwig Boltzmann Institute (O.P. and M.B.) at its premises. Animals had free access to food (rat chow V1534000, SSNIFF, Soest, Germany) and tap water. Preoperatively animals were assessed for appropriate hindlimb motor function using the Basso, Beattie, Bresnahan (BBB) locomotor rating scale.10 In case of discrepancies between raters, the lower score was accepted. The functional comparison between groups was assessed with BBB scores as given by 10.1021/pr060620k CCC: $37.00

 2007 American Chemical Society

PTMs of GDI-2 Indicate NO-Related and Oxidative Stress in SCI

Scheff.10 All animals revealed a preoperative BBB score of 21, thus, indicating homogeneity of the groups used. Surgical Interventions. Animals, 8 per group, were randomly assigned to sham controls and SCI groups (150 kdyn) prior to surgery and then anesthetized with intraperitoneal (ip) injections of 100 mg/kg ketamine hydrochloride (Ketalar; ParkeDavis, Berlin, Germany) and 10 mg/kg xylazine (Rompun, Bayer, Leverkusen, Germany). Atropine (Atropinum sulfuricum; Nycomed, Vienna, Austria) was given subcutaneously (0.3 mg/ kg). For maintenance of anesthesia, half of the initial ketamine/ xylazine/atropine dose was administered ip every 60 min. Core body temperature was measured with a rectal probe and maintained at 37-38 °C with a heating lamp until recovery. Each rat was subjected to laminectomy at the level of the 11th thoracic vertebra (T11). The laminectomy was only slightly larger than the 2.5 mm impactor tip of the contusion device. The exposed vertebral column was stabilized by clamping the rostral T10 and the caudal T12 with two forceps (Fine Science Tools, Heidelberg, Germany). Special attention was devoted to proper alignment of the spinal cord in a horizontal position, perpendicular to the impactor. SCI was induced with the Infinite Horizon impactor device (Precision Systems & Instrumentation, Lexington, KY).11 This device creates a reliable contusion injury to the spinal cord by applying a force-defined impact with an impounder. An in-line force sensor on the tip of the impounder measures the force applied by the motor rack to the specimen. A stepping motor, which is interfaced with an external computer, provides the force-controlled impact (150 kdyn). The correct conduction of the contusion (“hit”) was controlled on the microprocessor control unit, and data files were stored on hard disk. The surgical field was re-inspected, and the wound was closed in anatomical layers. Resorbable sutures were used for approximation of muscle and connective tissue (Ethicon, Norderstedt, Germany), and titanium staples were used for closure of the skin incision (Fine Science Tools, Heidelberg, Germany). Post-Surgery Animal Care. Subcutaneous (sc) injections of 8 mL Ringer’s solution and antibiotics (Peni-Strepto, 50 000 IU, Virbac Lab., Carros, France) were administered to all rats after surgery. Analgetic treatment was routinely supplied for 3 days postoperatively and consisted of intramuscular application of an antiphlogistic (Temgesic, 2 mg/kg body weight, Merck, Darmstadt, Germany) once daily. Animals were housed under simulated daylight conditions with alternating 12 h light and dark cycles. Standard rat chow and water was provided, and rats were checked daily for signs of infection or dehydration. If necessary, manual bladder expression was carried out until spontaneous urination occurred. Animals assigned to proteomic analysis were anaesthetized (as for surgery) and then euthanized by perfusion with 100 mL of PBS, containing 50 IU of heparin (Baxter, Deerfield, IL). This procedure was chosen in order not to chemically alter the tissue and allow consecutive proteomic analysis. The correct site of lesion was checked in all animals post-mortem by matching it to the 11th pair of ribs, the thoracic spinal cord was removed, and samples for proteomic analysis were immediately conserved in liquid nitrogen. Sample Preparation. Spinal cord tissue tissue was powderized and resuspended in 1.0 mL of sample buffer consisting of 7 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St. Louis, MO), 4% 3-[(3-cholamidopropyl) dimethylammonio]1-propane-sulfonate (CHAPS) (Sigma), 65 mM 1,4-dithioerythritol (Merck, Germany), 1 mM ethylenediaminetetraacetic acid (EDTA) (Merck), protease inhibitors complete (Roche, Basel,

research articles Switzerland), and 1 mM phenylmethylsulfonyl chloride. The suspension was sonicated for approximately 15 s. After homogenization, samples were left at room temperature for 1 h and centrifuged at 14 000g for 1 h. The supernatant was transferred into Ultrafree-4 centrifugal filter units (Millipore, Bedford, MA) for desalting and concentrating proteins.12 Protein content of the supernatant was determined by the Bradford protein assay system.13 The standard curve was generated using bovine serum albumin, and absorbance was measured at 595 nm. Two-Dimensional Gel Electrophoresis (2-DE). Protein extracts prepared from spinal cord were subjected to 2-DE as described elsewhere.14,15 Seven hundred micrograms of protein was applied on immobilized pH 3-10 nonlinear gradient strips at their basic and acidic ends. Focusing was started at 200 V, and voltage was gradually increased to 8000 V over 31 h and then kept constant for a further 3 h (approximately 150 000 V h totally). After the first dimension, strips (18 cm) were equilibrated for 15 min in the buffer containing 6 M urea, 20% glycerol, 2% SDS, 2% DTT, and then for 15 min in the same buffer containing 2.5% iodo-acetamide instead of DDT. After equilibration, strips were loaded on 9-16% gradient sodium dodecyl sulfate polyacrylamide gels for second-dimensional separation. Gels (180 mm × 200 mm × 1.5 mm) were run at 40 mA per gel. Immediately after the second-dimension run, gels were fixed for 18 h in 50% methanol, containing 10% acetic acid; the gels were then stained with Colloidal Coomassie Blue (Novex, San Diego, CA) for 12 h on a rocking shaker. Molecular masses were determined by running standard protein markers (Bio-Rad Laboratories, Hercules, CA) covering the range 10250 kDa. pI values of 3-10 were used as given by the supplier of the immobilized pH gradient strips (Amersham Bioscience, Uppsala, Sweden). Excess of dye was washed from the gels with distilled water, and the gels were scanned with ImageScanner (Amersham Bioscience). Electronic images of the gels were recorded using Adobe Photoshop and Microsoft Power Point Software. Method for MALDI-TOF/TOF Analysis. 1. Sample Preparation. Gel spots identified as GDI-2 in parallel experiments were excised manually and washed with 10 mM ammonium bicarbonate and 50% acetonitrile (ACN) in 10 mM ammonium bicarbonate. After washing, gel plugs were shrunk by addition of ACN and dried. The dried gel pieces were re-swollen with 40 ng/µL trypsin (Promega, Madison, WI) in digestion buffer (consisting of 5 mM octyl β-D-glucopyranoside (OGP) and 5 mM ammonium bicarbonate) and incubated for 4 h at 30 °C: Asp-N digests were performed by addition of 25 mM ammonium bicarbonate containing 25 ng/µL of Asp-N (sequencing grade; Roche Diagnostic, Mannheim, Germany) and incubated for 18 h at 31 °C. Extraction was performed with 10 µL of 1% TFA in 5 mM OGP. 2. Protein Identification by MALDI-TOF/TOF. Extracted peptides were directly applied onto a target (AnchorChipTM, Bruker Daltonics, Bremen, Germany) that was loaded with R-cyano-4-hydroxy-cinnamic acid (Bruker Daltonics) matrix thinlayer (saturated solution in 100% acetone with 0.1% TFA). The mass spectrometer used in this work was an UltraflexTM TOF/TOF (Bruker Daltonics) operated in positive-ion reflector mode for peptide mass analysis, and the “LIFT” mode was used for tandem mass spectrometry sequencing of peptides using the FlexControl software (Bruker Daltonics). An accelerating voltage of 25 kV was used for PMF. Calibration of the instrument was performed externally with [M + H]+ ions of angioJournal of Proteome Research • Vol. 6, No. 4, 2007 1501

research articles tensin I, angiotensin II, substance P, bombesin, and adrenocorticotropic hormones (clip 1-17 and clip 18-39). Each spectrum was produced by accumulating data from 200 consecutive laser shots for PMF. Those samples which were analyzed by PMF from MALDI-TOF were additionally analyzed using LIFT-TOF-TOF MS/MS from the same target using LID mode.16 In the LID-MS/MS mode using a long-lifetime N2 laser, all ions were accelerated to 8 kV under conditions promoting metastable fragmentation in the TOF1 stage. After selection of jointly migrating parent and fragment ions in a timed ion gate, ions were lifted by 19 kV to high potential energy in the LIFT cell. After further acceleration of the fragment ions in the second ion source, their masses could be simultaneously analyzed in the reflector with high sensitivity. PMF and MS/ MS spectra obtained from MALDI-TOF and MALDI-TOF/TOF were interpreted with the in-house licensed database search engine MASCOT software (version 2.1.04) (Matrix Science Ltd, London, U.K.). Database searches, through MASCOT, using combined PMF and MS/MS data sets were performed via BioTools 2.2 software (Bruker Daltonics). A mass tolerance of 25 ppm, MS/MS tolerance of 0.5 Da, and two missing cleavage site were allowed. The probability score calculated by the software was used as criterion for correct identification (http:// www.matrixscience.com/help/scoring_help.html). MS and MS/ MS spectra were searched against NCBI nucleic acid and SwissProt/TrEMBL Database. Oxidation of methionine was set as variable modifications, carbamidomethylation of cysteine residues as fixed modification. MASCOT results were confirmed manually. Unmatched MS/MS spectra were further analyzed by de novo sequencing analysis. MS/MS spectra were sequenced de novo using BioTools 2.2 software with RapidDeNovo extension (Bruker Daltonics16), and the top high-scoring candidate sequences for MS/MS spectra were then submitted to MS BLAST sequence similarity search (http://dove.emblheidelberg.de/Blast2/msblast.html), which was based on the most likely de novo sequences. Protein identification significance was judged using the MS BLAST scoring algorithm.17 Method for NanoESI-LC-MS/MS Analysis. 1. Sample Preparation. Gel spots were excised, cut into smaller pieces, and washed with ultrapure water. Coomassie-stained gel bands were destained by several washing steps with 20 mM ammonium bicarbonate and ACN (1:1). Gel pieces were dehydrated by adding ACN. After 5 min incubation, ACN was removed, and gel fragments were dried in the Speed-Vac. Dry gel pieces were incubated for 30 min at 56 °C in 200 µL of 10 mM dithiothreitol (DTT) to reduce disulfide bonds. DTT was washed off, and cysteines were alkylated by incubation with 100 µL of 54 mM iodoacetamide for 20 min at room temperature in the dark. After washing steps with 50 mM ammonium bicarbonate, the gel was dried and incubated for 4 h at 29 °C with 40 ng/µL trypsin (Promega, Madison, WI) in 50 mM ammonium carbonate buffer, pH 8.5; Asp-N digests were performed by addition of 25 mM ammonium bicarbonate containing 25 ng/µL of Asp-N (sequencing grade; Roche Diagnostic, Mannheim, Germany) and incubated for 18 h at 31 °C. The reaction was stopped by adding 10% FA to a final concentration of ∼1%. After 10 min of sonication the supernatant was taken off and peptides were further extracted by adding approximately 20 µL of 5% formic acid (FA) followed by 10 min sonication (procedure was repeated once). Protein Identification by Nano-ESI-LC-MS/MS. 2. Analysis of Peptides by Nano-LC-MS/MS. The peptide mixture obtained from tryptic digests was separated by Ultimate 3000 1502

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nanoLC system (Dionex) and analyzed using QSTAR XL (Applied Biosystems, Foster City, CA) equipped with a nano electrospray ionization source. For nano-ESI-LC-MS/MS, the digest was loaded onto PepMap 100 C18 precolumn (300 µm i.d., 5 mm long cartridge, from Dionex, Amsterdam, Netherlands) from 0 to 30 min and then separated by PepMap 100 C18 analytic column (75µ m i.d., 150 mm long cartridge, from Dionex) using a linear gradient of 4% B (Solvent A: 0.1% FA; Solvent B: 80% ACN/0.08% FA) to 60% from 0 to 30 min, 90% B constant from 30 to 35 min, and 4% B from 35 to 60 min using the Ultimate micropump at a flow rate of 300 nL/min. As peptides eluted from LC, they were electrosprayed into QSTAR XL. Each cycle consisted of one full scan mass spectrum (m/z 350-1600) followed by MS/MS spectra on the three most abundant peptide ions in the full MS scan. The derived mass spectrometry data sets were converted to MASCOT generic format flat files by macot.dll (Matrix Science, Boston, MA) script supplied with AnalystQS software (Applied Biosystems) and searched against in-house licensed MSDB 20051115 and Expasy/TrEMBL databases.18 Oxidation of methionine was set as variable modifications, carbamidomethylation of cysteine residues as fixed modification. The PHENYX software platform (version 2.0) from GenBio (Geneva, Switzerland) was used to confirm the peptide identification and modification. The raw MS/MS data were converted to .mgf file and submitted to PHENYX. Database searches were carried out against an in-house built Swiss-Prot/ UniProt database. Top-scoring candidate peptides were manually validated with MASCOT results. Bioinformatics Analysis. Protein sequences derived from NCBI Protein Database were used for protein sequence analysis. ClustalW (http://www.ebi.ac.uk/clustalw/) was used for multiple sequence alignment searches. ELM (http://elm.eu.org) was used for functional domain identification. Protein Quantification. Protein spots from 32 gels (8 per group) were outlined (first automatically and then manually) and quantified using the Proteomweaver software (Definiens, Munich, Germany).19 The percentage of the volume of the spots representing a certain protein was determined in comparison with the total proteins present in the 2-DE gel. Values are expressed as means ( standard deviation (SD) of percentage of the spot volume in each particular gel after subtraction of the background values. Student’s t test was applied, and the level of significance was set at p < 0.05.

Results Differential Expression of GDI-2 in SCI Compared with Sham-Operated Control. To investigate alterations in GDI-2 protein levels associated with SCI in a time-dependent manner, we performed comparative high-resolution, two-dimensional gel electrophoresis experiments on protein extracts obtained from 8 and 72 h post-injury and sham-operated controls. Spinal cord extracts were resolved using immobilized pH gradient strips followed by separation on SDS-PAGE. Total proteins in the two-dimensional gels were visualized by staining with colloidal Coomassie blue. Gel images from sham-operated controls and samples obtained from different time points (8 and 72 h) were compared with Proteomweaver quantification software to determine differential protein levels. Quantification of the intensities of both sham-operated control and samples of 8 h post-injury revealed that in the 8 h of post-injury samples, GDI-2 protein levels were increased 3-fold as compared with those in the sham control (p < 0.0045) (Figure 1,


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Figure 1. (a) Two-dimensional gel images of GDI-2 protein levels in SCI and control. Representative two-dimensional gels (A and B) of control (A) and 8 h following injury sample (B) are displayed left and right, respectively. GDI-2 protein spots are marked in box. The corresponding protein spots were identified by mass spectrometry. Enlarged images of the 2-D of the control (C) and 8 h following injury (D) sample are shown for GDI-2. Three-dimensional visualization of Control (E) and 8 h of SCI (F) indicate GDI-2 up-regulation in SCI. (b) Differential expression of GDI-2 in sham control and 8 h of post-injury sample. The intensity of protein spots indicated in panel a was measured using two-dimensional gel analysis program Proteomeweaver. Total protein level significantly increased to 3-fold compared to sham control. The asterisk indicates statistically significant (p < 0.0045) difference in the protein level of GDI-2 protein spot in SCI transected versus control. Journal of Proteome Research • Vol. 6, No. 4, 2007 1503

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Figure 2. Identification of up-regulated protein spot as GDI-2 by MALDI-TOF/TOF and nano-ESI-LC-MS/MS. (a) MS spectrum from tryptic cleavage of GDI-2 and MS/MS spectrum from peptide 194TDDYLDQPCCETINR208 with m/z of 1899.8730 analyzed with MALDITOF/TOF are shown. The asterisk indicates that the cystine is modified with carbamidomethyl. (b) MS/MS spectrum of the ion at m/z 1807,287 from tryptic cleavage of GDI-2, and amino acid sequence 349YIAIVSTTVETKEPEK364 analyzed with nanoESI-LC-MS/MS was unambiguously assigned to GDI-2. 1504


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Table 1. Measured and Calculated Protonated (M + H) Molecular Masses and Sequences of Peptides Determined from GDI-2 by MALDI-TOF/TOFa Mr

a

start-end

experimental

calculated

delta

missb

sequencec

30-54 56-68 56-68 69-79 90-98 90-98 143-153 143-156 174-193 175-193 194-208 222-240 241-264 241-264 310-328 329-348 365-379 380-390 391-402 403-422 424-436 424-436

2855.2442 1384.7120 1400.7050 1311.6934 1108.5872 1124.5707 1343.6653 1711.8335 2294.1136 2166.0148 1898.7397 2140.0348 2650.3065 2666.3194 2197.9735 2238.0613 1776.9554 1250.6858 1350.6696 2406.0523 1605.6558 1621.6595

2855.3323 1384.7285 1400.7234 1311.6935 1108.5950 1124.5900 1343.6761 1711.8569 2294.1695 2166.0745 1898.7775 2140.0992 2650.4039 2666.3988 2198.0498 2238.0925 1776.9985 1250.6910 1350.6779 2406.0685 1605.6803 1621.6752

-0.0881 -0.0165 -0.0184 -0.0001 -0.0078 -0.0192 -0.0108 -0.0234 -0.0558 -0.0597 -0.0377 -0.0645 -0.0974 -0.0794 -0.0763 -0.0312 -0.0431 -0.0052 -0.0083 -0.0162 -0.0245 -0.0156

0 1 1 1 0 0 0 1 1 0 0 0 0 0 0 1 0 0 0 1 1 1

VLHM*DQNPYYGGESASITPLEDLYK.R FKLPGQPPASMGR FKLPGQPPASM*GR GRDWNVDLIPK MLLFTEVTR M*LLFTEVTR FLVYVANFDEK FLVYVANFDEKDPR KFDLGQDVIDFTGHSLALYR FDLGQDVIDFTGHSLALYR TDDYLDQPCCETINR SPYLYPLYGLGELPQGFAR LSAIYGGTYMLNKPIEEIIVQNGK LSAIYGGTYM*LNKPIEEIIVQNGK NTNDANSCQIIIPQNQVNR KSDIYVCMISFAHNVAAQGK EIRPALELLEPIEQK FVSISDLFVPK DLGTDSQIFISR AYDATTHFETTCDDIKDIYK MTGSEFDFEEMKR MTGSEFDFEEMKR

Letters marked in bold letters represent data obtained from MALDI/TOF/TOF or with MS/MS analysis. b Missed cleavage number. c M*, oxidized methionine.

panels a and b). At 72 h post-injury, however, GDI-2 protein levels were not significantly different (data not shown). Identification of Protein Spots by Mass Spectrometry. For determination of the identity of GDI-2, the spot was excised from two-dimensional electrophoresis gels and digested with trypsin. The masses of the tryptic peptide fragments were measured using MALDI-TOF/TOF and nano-ESI-LC-MS/MS analysis. Observed ions were searched with MASCOT against Expasy/TrEMBL (http://www.expasy.org) and NCBI Protein Database (http://www.ncbi.nlm.nih.gov). Peptide mapping and MS/MS measurements on tryptic fragments led to confirmation of rat GDP-dissociation inhibitor 2 (GDI-2) (NCBI protein database accession no: NP_058972). Figure 2 and Table 1 summarize the peptide sequence determined by MS and MS/ MS analysis of GDI-2. This assignment was further strengthened by the agreement between the apparent molecular mass/ pI values of 49.5 kDa/6.1 of GDI-2 and the predicted values of 50.5 kDa/5.93. There were two rat GDI-2 protein sequences (GDP dissociation inhibitor 2, NP_058972; Rab GDI beta, P50399) given in the NCBI protein database, exhibiting 98% sequence similarity to each other. Both rat GDI-2 protein sequences were different from each other at amino acid numbers 205-211 and 231232 (see Supporting Information Figure 1). MS/MS analysis of the tryptic-digested peptides [M + H]+ at m/z 1898.7397, m/z 2140.0348. and their corresponding sequences, TDDYLDQPCCETINR (amino acid residues 194-208), SPYLYPLYGLGELPQGFAR (amino acid residues 222-240), were perfectly matched to GDP dissociation inhibitor 2 (NP_058972) with high score (Figure 2a, Table 1). This result confirmed that rat GDP dissociation inhibitor 2 and rat Rab GDI beta represent two different isoforms. Molecular Characterization of Protein Modifications in SCI. 1. Protein Nitration at Tyrosine Y-224. To examine whether GDI-2 was oxidized or nitrated in SCI, individual protein spots from different time points and sham-operated controls were excised and digested with trypsin and Asp-N. Individual

digested peptides were analyzed by MALDI-TOF/TOF and nanoESI-LC-MS/MS. Protein sequences were determined using MASCOT and de novo sequencing methods. Ninety percent of sequence coverage was reached when peptides of all GDI-2 spots were considered using both mass spectrometric approaches. MS/MS experiments alone led to sequence coverage of 88% (see Supporting Information Figure 2). The minimal sequence coverage of all GDI-2 spots was at least 75% of GDI-2 from all sham-operated controls and different time points of SCI (see Supporting Information Tables 1-3). In contrast to the findings in sham-operated rats and later time points, at 8 h post-injury, the peptide 222SPYLYPLYGLGELPQGFAR240 contained 3-aminotyrosine as shown by MALDITOF analysis (Figure 3). Comparing to the unmodified peptide mass (m/z 2141.19), the modified peptide mass (m/z 2156.18) was increased to 15 Da. To obtain specific identification of the 3-aminotyrosine modification site, further analysis with MS/ MS experiments and subsequent analysis with MASCOT confirmed that Y-224 was modified to 3-aminotyrosine with high score (ion score 70). This modification was further verified by the observation of complete side chain loss of 3-aminotyrosine at m/z 2034.25, that is, m/z 2156.14 - 122, m/z 2034.25, as evaluated by MALDI-TOF/TOF (Figure 4a). In addition, a series of fragments of ions corresponding to y-ion series (y1-y16) and internal ions fragments were observed. The mass of the b2-ion at m/z 185.005 (di-peptide mass) and y16-ion at m/z 1793.96 was unchanged as compared to that of the unmodified peptide MS/MS data. This result indicates that only one possibility exists that the mass corresponding to Y-224 increased to 15 Da. Taken together, these data allowed assignment of 3-aminotyrosine to Y-224 (Figure 4b). 2. Protein Oxidation: Modification of K-174. MALDI/TOF analysis of the tryptic peptide obtained from 72 h post-injury samples indicated that the observed ion at m/z 2376.13 was not found in sham-operated controls nor at 8 h after injury. Further MS/MS and de novo sequencing analyses were carried Journal of Proteome Research • Vol. 6, No. 4, 2007 1505

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Figure 3. Identification of the nitrated site by MALDI/MS. Spectra of nitrated fragments of GDI-2. The mass ion of the unmodified tryptic fragment and of its reduced product (Y-NH2) is indicated. When compared to sham-operated control, a peptide modified with 3-aminotyrosine (ion at m/z 2156.168) was only observed in 8 h of post-injury sample. The asterisk indicates the unmodified peptide mass.

out: the MS/MS spectrum of ion at m/z 2376.13 showed a series of b- and y-ions. The sequence was derived from the spectrum based on complementary b- and y-ions information. De novo sequencing analysis confirmed the sequence 174KFDLGQDVIDFTGHSLALYR193 as the mass was increased by 81 Da compared with the original peptide mass (m/z 2294.17) (Figure 5a). Moreover, the b1-ion was observed at m/z 210.054 corresponding to a mass increase of 81 Da in the N-terminal lysine (K-174) residue (Figure 5b). This mass elevation is considered to result from the modification of the N-terminal lysine residue by both carbonyl adducts, acrolein (+38 Da) and carbamyl (+43 Da). 3. Domain Analysis. The ELM database (http://elm.eu.org) revealed the presence of different motifs including the PxLxP motif between amino acids 221 and 228 and the Src-family SRC-Homology 2 (SH2) domain binding motif at 224-227.

Discussion The major outcome of the study is to show that nitration, as well as oxidation, occurs during the course of SCI at specific and different time points. Tyrosine nitration as represented by aminotyrosine was observed at 8 h following the traumatic 1506


lesion, and oxidation expressed as acrolein and carbamyl adducts of lysine was detected at 72 h post-injury exclusively. SCI generates reactive nitrogen species causing secondary damage by nitrating proteins.19,20 Peroxynitrite reacts with protein-tyrosine (P-Y) residues to form 3-nitrotyrosine (YNO2). This modification has been detected in SCI but is not the only product. Working under reducing conditions as, for example, using reactive thiols in two-dimensional gel electrophoresis, leads to reduction of 3-nitrotyrosine to 3-aminotyrosine (Y-NH2), and this has to be considered when tyrosine nitration is being evaluated.8 An HPLC method and an MS technique has been developed for the detection of 3-aminotyrosine,21,22 and it was shown that this product was increased in amyotrophic lateral sclerosis.9 Herein, this modification in a GDI-2 peptide was detected by MALDI-MS/MS in a sitespecific manner, that is, on tyrosine 224. This work, therefore, provides complementary and more specific information to that obtained by detection of the product in hydrolysates per se. When MALDI-MS/MS analysis is used, the position in the sequence can be determined, and indeed, Y-224 is an amino acid present in two functional domains/motifs of GDI-2, and the modification may well lead to impairment of GDI-2


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Figure 4. Identification of the nitrated sites in GDI-2 by MALDI-TOF/TOF analysis. (a) Represents an enlarged view of the signal at m/z 2035.58 which corresponds to a complete loss of the 3-aminotyrosine side chain from the precursor ion. (b) MALDI-TOF/TOF spectrum of the ion at m/z 2156.1880 from tryptic digests of GDI-2 after 8 h of post-injury showing Y-224 modified with aminotyrosine. Journal of Proteome Research • Vol. 6, No. 4, 2007 1507

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Figure 5. Identification of oxidation site in GDI-2 by MALDI-TOF/TOF analysis. MALDI-TOF/TOF spectrum of ion at m/z 2376.17 from the tryptic digest of GDI-2 after 72 h of SCI showing that the mass of b1-ion increased to 81 Da corresponding to K-174 modified with both, acrolein and cabamylation. The asterisk indicates that the N-terminal lysine residue was modified with acrolein and carbamylation.

function that remains to be evaluated in further functional studies. The motif PxLxP/MYND ligand is a zinc finger observed in certain transcription factors (http://elm.eu.org/elmPages/ LIG_MYND.html), and the modified tyrosine residue could inhibit function of this motif. Modification of the second motif, Src Homology 2 (SH2), may well be interfering with the widely accepted and well-documented signalling activity of this structure (http://elm.eu.org/elmPages/LIG_SH2_SRC.html). As shown by mutational analysis changes in the sequence do lead to impairment of binding to proteins.23 During the lipid peroxidation process, decomposition of lipid hydroperoxides leads to generation of many compounds as reactive intermediates that may bind to amino acid residues of protein, generating relatively stable end products. Among all lipid peroxidation byproducts (R, β-unsaturated aldehydes), acrolein is the most active toward nucleophiles such as lysine.24 Because of oxidative stress, acrolein undergoes nucleophilic addition at the double bond (C-3) to form a secondary derivative with retention of the aldehyde group, resulting in the formation of Michael addition-type acrolein-lysine adducts.24 Protein-bound acrolein is an important marker of oxidative stress.25 Formation of acrolein in a guinea pig model of SCI model after 24 h post-injury was described,5 and carbamylation was observed of the same lysine residue. While carbamylation may be due to technical reasons as urea used, carbamylation was only observed along with the acrolein adduct, suggesting that acrolein may increase susceptibility of the protein toward carbamylation, probably due to increased nucleophilicity. The acrolein adduct of lysine may well be 1508


interfering with binding as proposed above for nitration of tyrosine as domain/motif function may be impaired by the modification. Taken together, two mass spectrometrical methods were applied to examine oxidation and nitration-related modifications of GDI-2 protein. While nanoESI-LC-MS/MS added significantly to the identification of the protein, peptides and their modifications detected by MALDI-TOF/TOF could not be revealed by this method. MALDI-MS/MS may therefore represent complementary tools for the detection of oxidation or nitration-related modifications. The 3-fold increase of GDI-2 protein in SCI at 8 h following the insult may be of relevance for signalling, as may be modification of the domain(s) that may be inhibiting binding. Last, but not least, we propose the use of the described analytical tools to determine site-specific modification in SCI and propose to carry out analysis of 3-aminotyrosine to avoid misinterpretations if 3-nitrotyrosine is determined exclusively. We are currently using GDI-2 protein as a probe to test post-translational modifications occurring during the clinical course of SCI in the rat to obtain clues for pathogenesis and to search for pharmacological targets.

Acknowledgment. We acknowledge the technical contribution by Nadja Walder, Mika Brenjikow, Rudolf Hopf and Angela Schneider. Supporting Information Available: Table listing the measured and calculated molecular masses and sequences of peptides determined from GDI-2 after 8 and 72 h of impact and in controls using MS; figures showing the comparison of protein sequence of rat GDI-2 and mouse and rat Rab GDI

research articles


beta-2; the amino acid sequence of Rat GDI-2 from rats as obtained from chemical sequence analysis generated by trypsin and Asp-N digest. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Jesberger, J. A.; Richardson, J. S. Oxygen free radicals and brain dysfunction. Int. J. Neurosci. 1991, 57, 1-17. (2) Stadman, E. R.; Oliver, C. N. Metal-catalyzed oxidation of proteins. Physiological consequences. J. Biol. Chem. 1991, 266, 2005-2008. (3) Leski, M. L.; Bao, F.; Wu, L.; Qian, H.; Sun, D.; Liu, D. Protein and DNA oxidation in spinal injury: neurofilamentssan oxidation target. Free Radical Biol. Med. 2001, 30, 613-624. (4) Aksenova, M.; Butterfield, D. A.; Zhang, S. X.; Underwood, M.; Geddes, J. W. Increased protein oxidation and decreased creatine kinase BB expression and activity after spinal cord contusion injury. J. Neurotrauma 2002, 19, 491-502. (5) Luo, J.; Uchida, K.; Shi, R. Accumulation of acrolein-protein adducts after traumatic spinal cord injury. Neurochem. Res. 2005, 30, 2951-2955. (6) Liu, D.; Ling, X.; Wen, J.; Liu J. The role of reactive nitrogen species in secondary spinal cord injury: formation of nitric oxide, peroxynitrite, and nitrated protein. J. Neurochem. 2000, 75, 21442154. (7) Bao, F.; DeWitt, D. S.; Prough, D. S.; Liu D. Peroxynitrite generated in the rat spinal cord induces oxidation and nitration of proteins: reduction by Mn (III) tetrakis (4-benzoic acid) porphyrin. J. Neurosci. Res. 2003, 71, 220-227. (8) Balabanli, B.; Kamisaki, Y.; Martin, E.; Murad, F. Requirements for heme and thiols for the nonenzymatic modification of nitrotyrosine. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13136-13141. (9) Casoni, F.; Basso, M.; Massignan, T.; Gianazza, E.; Cheroni, C.; Salmona, M.; Bendotti, C.; Bonetto, V. Protein nitration in a mouse model of familial amyotrophic lateral sclerosis: possible multifunctional role in the pathogenesis. J. Biol. Chem. 2005, 280, 16295-16304. (10) Basso, D. M.; Beattie, M. S.; Bresnahan, J. C. A sensitive and reliable locomotor rating scale for open field testing in rats. J. Neurotrauma 1995, 12, 1-21. (11) Rabchevsky, A. G.; Sullivan, P. G.; Fugaccia, I.; Scheff, S. W. Creatine diet supplement for spinal cord injury: influences on functional recovery and tissue sparing in rats. J. Neurotrauma 2003, 20, 659-669. (12) Weitzdorfer, R.; Hoger, H.; Pollak, A.; Lubec, G. Changes of hippocampal protein levels during postnatal brain development in the rat. J. Proteome Res. 2006, 5, 3205-3212. (13) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. (14) Weitzdoerfer, R.; Fountoulakis, M.; Lubec, G. Reduction of actinrelated protein complex 2/3 in fetal Down syndrome brain. Biochem. Biophys. Res. Commun. 2002, 293, 836-841. (15) Yang, J. W.; Czech, T.; Lubec, G. Proteomic profiling of human hippocampus Electrophoresis 2004, 25, 1169-1174.

(16) Suckau, D.; Resemann, A.; Schuerenberg, M.; Hufnagel, P.; Franzen, J.; Holle A. A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Anal. Bioanal. Chem. 2003, 376, 952-965. (17) Shevchenko, A.; Sunyaev, S.; Loboda, A.; Shevchenko, A.; Bork, P.; Ens, W.; Standing, K. G. Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time-of-flight mass spectrometry and BLAST homology searching. Anal. Chem. 2001, 73, 1917-1926. (18) Chen, W. Q.; Kang, S. U.; Lubec, G. Protein profiling by the combination of two independent mass spectrometry techniques. Nat. Protocols 2006, 1, 1446-1452. (19) Challapalli, K. K.; Zabel, C.; Schuchhardt, J.; Kaindl, A. M.; Klose, J.; Herzel, H. High reproducibility of large-gel two-dimensional electrophoresis. Electrophoresis 2004, 25, 3040-3047. (20) Kanski, J.; Alterman, M. A.; Schoneich, C. Proteomic identification of age-dependent protein nitration in rat skeletal muscle. Free Radical Biol. Med. 2003, 15, 1229-1239. (21) Halliwell, B. Oxidants and the central nervous system: some fundamental questions. Is oxidant damage relevant to Parkinson’s disease, Alzheimer’s disease, traumatic injury or stroke? Acta Neurol. Scand. Suppl. 1989, 126, 23-33. (22) Halliwell, B. Oxidants and human disease: some new concepts. FASEB J. 1987, 1, 358-364. (23) Beckman, J. S.; Beckman T. W.; Chen, J.; Marshall, P. A.; Freeman, B. A. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1620-1624. (24) Saran, M.; Michel, C.; Bors, W. Reaction of NO with O2implications for the action of endothelium-derived relaxing factor (EDRF). Free Radical Res. Commun. 1990, 10, 221-226. (25) Sarver, A.; Scheffler, N. K.; Shetlar, M. D.; Gibson, B. W. Analysis of peptides and proteins containing nitrotyrosine by matrixassisted laser desorption/ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 2001, 12, 439-448. (26) Ohshima, H.; Celan, I.; Chazotte, L.; Pignatelli, B.; Mower, H. F. Analysis of 3-nitrotyrosine in biological fluids and protein hydrolyzates by high-performance liquid chromatography using a postseparation, on-line reduction column and electrochemical detection: results with various nitrating agents. Nitric Oxide 1999, 3, 132-141. (27) Shalk, I.; Zeng, K.; Wu, S.; Stura E. A.; Matteson, J.; Huang, M.; Tandon, A.; Wilson, I. A.; Balch, W. E. Structure and mutational analysis of Rab GDP-dissociation inhibitor. Nature 1996, 381, 4248. (28) Uchida, K.; Kanematsu, M.; Sakai, K.; Matsuda., T.; Hattori, N.; Mizuno, Y.; Suzuki, D.; Miyata, T.; Noguchi, N.; Niki, E.; Osawa, T. Protein-bound acrolein: potential markers for oxidative stress. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4882-4887. (29) Uchida, K.; Kanematsu, M.; Morimitsu, Y.; Osawa, T.; Noguchi, N.; Niki, E. Acrolein is a product of lipid peroxidation reaction. Formation of free acrolein and its conjugate with lysine residues in oxidized low density lipoproteins. J. Biol. Chem. 1998, 273, 16058-16066.

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Journal of Proteome Research • Vol. 6, No. 4, 2007 1509

in Spinal Cord Injury of the Rat - American Chemical Society

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