3006 Julius Paul Pradeep John Arnold Pollak Gert Lubec Department of Pediatrics, Medical University of Vienna, Vienna, Austria
Received March 12, 2009 Revised May 18, 2009 Accepted May 21, 2009
Electrophoresis 2009, 30, 3006–3016
Research Article
Complete sequencing and oxidative modification of manganese superoxide dismutase in medulloblastoma cells Manganese superoxide dismutase (Mn-SOD or SOD2) is a key antioxidant enzyme and was assigned several roles in tumor biology. Working on medulloblastoma cell line DAOY, we identified two spots as Mn-SODs. Because of the proposed pivotal role of this enzyme in oncobiology, we decided to completely sequence the proteins and to determine PTMs. Proteins extracted from DAOY cells were run on 2-DE, multienzyme digestions were carried out and peptides were analyzed by MALDI-TOF/TOF, Qq-TOF and the ion trap using both the CID and ETD principles. Both protein expression forms were completely sequenced and revealed identical protein sequences. Histidines His30 and His31 were oxidized in one protein, whereas tryptophan oxidation (Trp-186) was observed in both. Histidine oxidation was not only indicated by the mass shift of the peptide but also by specific spectra of 2-oxo-histidine and a previously described intermediate (His114). Complete sequencing of the two Mn-SOD expression forms unambiguously characterizes this enzyme from a tumor cell line providing evidence that can be used for generation of antibodies and allowing conformational studies. The findings of different PTMs in the same gel represent Mn-SOD oxidative states, while oxidative modification of His30 and 31 may even reflect decreased Mn-SOD activity. Keywords: 2-Oxo-histidine / Manganese superoxide dismutase / Medulloblastoma / Proteomics / Reactive oxygen species DOI 10.1002/elps.200900168
1 Introduction Reactive oxygen species (ROS) such as superoxide (O2 ) and hydrogen peroxide (H2O2), are constantly produced during normal metabolic process in all living organism. ROS have mulitifactorial effects on the regulation of cell growth and proliferation [1, 2]. However, excessive ROS accumulation can cause cellular injury such as damage to DNA [3, 4], protein [5] and lipid membranes [6]. Due to their harmful effect, excessive ROS must be eliminated by a variety of antioxidant enzymes such as superoxide dismutases, catalases and various peroxidases. The cytosolic copper/ zinc-containing superoxide dismutase (Cu, Zn-SOD or
Correspondence: Professor Gert Lubec, Medical University of Vienna, Department of Pediatrics, Wa¨hringer Gu¨rtel 18-20, 1090 Vienna, Austria E-mail:
[email protected] Fax: 143-1-40400-6065
Abbreviations: HCT, high-capacity ion trap; Mn-SOD or SOD2, manganese superoxide dismutase; ROS, reactive oxygen species; Trp, tryptophan
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
SOD1) and the mitochondrial manganese superoxide dismutase (Mn-SOD or SOD2) are major enzymes responsible for catalyzing the conversion of superoxide to hydrogen peroxide, which is further removed by catalase and peroxidase [7]. Mn-SOD plays an important role in maintaining the cellular ROS balance in mitochondria, the major site of superoxide generation. Mn-SOD is a homotetramer, which contains one manganese ion per subunit [8]. The Mn-SOD gene is located on chromosome 6q25 [9]. Genetic inactivation of the Mn-SOD gene in mice results in the formation of neurological impairment and cardiotoxicity [10]. Heterozygous mice with lowered Mn-SOD activity reveal increased mitochondrial oxidatitive damage [11, 12]. High Mn-SOD activities are observed in heart, brain, liver and kidney [13]. A number of Mn-SOD variants with possible effects in cancer development have been described [14]. Keller et al. have shown that over expression of Mn-SOD in pheochromocytoma PC6 cells prevents neural apoptosis [15]. Church et al., reported that induction of Mn-SOD in melanoma cells reduces tumorigenicity in-vivo [16]. Ye et al., identified this protein as a metastasis marker for oral squamous cell carcinoma using proteomics-based www.electrophoresis-journal.com
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methodology and immunohistochemistry [17]. In medulloblastoma tissue in contrast to normal brain specimen, Mn-SOD is highly expressed [18]. Medulloblastoma is the most frequent malignant brain tumor in children and is considered to be of neuroectodermal origin. Carrying out proteomic studies [19] on the human medulloblastoma cell line DAOY, we observed two spots of Mn-SOD on 2-DE and because of the importance of this antioxidant protein in tumor biology we decided to completely sequence the two spots representing Mn-SOD and, in addition, to search for PTMs. Studies on these two expression forms in tumors were not reported in literature before, although Yin et al., detected two spots for the Mn-SOD protein using global proteomics analysis in embryonic stem cell-derived smooth muscle cells. These authors identified two spots by partially sequencing using proteomics methods and no PTMs were described [20]. Studies on PTMs are, however, mandatory and may be expected as under various conditions of oxidative stress, PTMs of Mn-SOD may occur resulting into decrease in MnSOD activity [21]. In addition, Mn-SOD has been shown to be nitrated by peroxynitrite with remarkable decrease in its enzymatic activity [22, 23]. Herein we report 100% sequence coverage of both proteins demonstrating the identical protein sequence of Mn-SOD that is different from other known Mn-SODs in databases. They differ, however, in terms of oxidation: spot one does not reveal oxidation of histidine 30 and 31, whereas tryptophan oxidation (Trp-186) was observed in both proteins. Characterization of the expression forms and PTMs may be of importance for design of further studies and interpretation of previous studies on Mn-SOD in tumor biology.
2 Materials and methods 2.1 Cell culture DAOY cells were kindly provided by Dr. M. Grotzer, grown and harvested as described previously [19]. Cells were scraped from dishes, washed three times in 2 mL of icecold phosphate-buffered saline (Gibco BRL, Gaithersburg, MD, USA), and centrifuged 3 min at 41C and 1000 g. The cell pellet was snap-frozen in liquid nitrogen and stored at 801C; the freezing chain was never interrupted.
2.2 Sample preparation The cell pellet was suspended in 1 mL of sample buffer consisting of 7 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St. Louis, MO, USA), 4% CHAPS (Sigma), 65 mM 1,4-dithioerythritol (Merck), 1 mM EDTA (Merck), protease inhibitors complete (Roche Diagnostic, Basel, Switzerland) and 1 mM phenylmethylsulfonyl chloride. The suspension was sonicated for approximately 15 s on & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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ice. After homogenization, samples were left at 211C for 1 h and centrifuged at 14 000 g for 1 h. The supernatant was transferred into Ultrafree-4 centrifugal filter units (Millipore, Bedford, MA, USA) for desalting and concentrating proteins. The protein content of supernatants was quantified by the Bradford protein assay system [24]. The standard curve was generated using bovine serum albumin, and absorbance was measured at 595 nm.
2.3 2-DE Samples prepared from the cell line were subjected to 2-DE as described elsewhere [25]. One milligram 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 Vh totally). After the first dimension, strips (18 cm) were equilibrated for 15 min in the buffer containing 6 M urea, 20% glycerol, 2% SDS and 2% DTT and subsequently for 15 min in the same buffer containing 2.5% iodo-acetamide instead of DTT. After equilibration, strips were loaded on 9–16% gradient sodium dodecylsulfate 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; gels were then stained with Colloidal Coomassie Blue (Novex, San Diego, CA, USA) for 12 h on a rocking shaker. Molecular masses were determined by running standard protein markers (Bio-Rad Laboratories, Hercules, CA, USA) covering the range 10–250 kDa. The pI values 3–10 were used as given by the supplier of the immobilized pH gradient strips (Amersham Bioscience, Uppsala, Sweden). Excess of dye was washed out from gels with distilled water, and gels were scanned with ImageScanner (Amersham Bioscience). Electronic images of the gels were recorded using Adobe Photoshop.
2.4 MALDI-TOF/TOF analysis 2.4.1 Sample preparation Gel spots identified as Mn-SOD in parallel experiments by MALDI-TOF/TOF were excised manually and washed with 10 mM ammonium bicarbonate and 50% 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/mL trypsin (Promega, Madison, WI, USAs) in digestion buffer (consisting of 5 mM octyl b-D-glucopyranoside and 5 mM ammonium bicarbonate) and incubated for 4 h at 301C: Asp-N digests were performed by addition of 25 mM ammonium bicarbonate containing 25 ng/mL of Asp-N (sequencing grade; Roche Diagnostic, Mannheim, Germany) and incubated for 18 h at www.electrophoresis-journal.com
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311C. Chymotrypsin digestions were performed by addition of 25 mM ammonium bicarbonate containing 25 ng/mL chymotrypsin (sequencing grade; Roche Diagnostic) and incubated for 2.5 h at 301C. Extraction was performed with 10 mL of 1% TFA in 5 mM octyl b-D-glucopyranoside.
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Mixtures were stirred and allowed to react for overnight at 211C. White crystal products were washed with hexane and dried in vacuum.
2.6 In-gel labeling and proteolytic digestions 2.4.2 Protein identification by MALDI-TOF/TOF Extracted peptides were directly applied onto a target (AnchorChipTM, Bruker Daltonics, Bremen, Germany) that was loaded with a-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 MS/MS sequencing of peptides using the FlexControlTM 2.4 software (Bruker Daltonics). An accelerating voltage of 25 kV was used for PMF. Calibration of the instrument was performed externally with [M1H]1- ions of angiotensin I, angiotensin II, substance P, bombesin and adrenocorticotropic hormones (clips 1–17 and clips 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 laser-induced dissociation mode [25]. In the laser-induced dissociation-MS/MS mode using a longlifetime 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. Mass spectra were analyzed using the Flex Analysis 2.4 software. PMF and MS/MS data sets were interpreted with MASCOT (Matrix Science, London, UK) software searched against MSDB 20051115 database (ftp://ftp.ncbi.nih.gov/repository/MSDB/) via BioTools 2.2s software (Bruker Daltonics). A mass tolerance of 25 ppm and MS/MS tolerance of 0.2 Da and one missing cleavage site were allowed. The probability score calculated by the software was used as criterion for correct identification. Oxidation of methionine, deamidation of Asn and Gln residues were set as variable modifications, carbamidomethylation of cysteine residues as fixed modification. MASCOT results were confirmed manually.
2.5 Synthesis of N-acetoxy- (1H3) succinimide N-hydroxysuccinimide (Sigma-Aldrich), and acetic anhydride (499% purity, Sigma-Aldrich) were used as purchased. N-acetoxy- (1H3) succinimide was synthesized as described previously [26]. Briefly, 4 g of N-hydroxysuccinimide were mixed with either 19.8 mL of acetic anhydride. & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Mn-SOD spots from 2-DE gels were excised for labeling reactions. Gel pieces were washed with 50% ACN and dried in a SpeedVac concentrator. Cysteine residues of proteins were reduced with 10 mL of 10 mM DTT at 561C for 30 min and alkylated with 10 mL of 55 mM iodoacetamide for 45 min at 211C. Gel pieces were then washed two times with 150 mL of 50% ACN and dried in a SpeedVac. Approximately 6 mg of N-acetoxy- (1H3) succinimide were added to dried gel pieces of Mn-SOD protein spots. In total 50 mL of 50 mM HEPES (pH 8.3) were added to each gel piece, vortexed for 1 min to dissolve and subsequently spun down in a microcentrifuge. Reactions were allowed to incubate for 3 h at 211C. The gel pieces were washed with 170 mL of 100 mM ammonium bicarbonate followed by 20 mL of 50% hydroxylamine solution for 20 min and incubated at 211C. Labeled gel slices were then washed three times with 150 mL of 100 mM ammonium bicarbonate and two times with 50 mL of ACN prior to drying in a SpeedVac. In total 12 mL of 40 ng/mL modified trypsin (sequencing grade; Promega) was added to the combined gel pieces and placed on ice for 15 min to swell the gel pieces with enzyme solution. An additional 45 mL of 50 mM ammonium bicarbonate (pH 8.4) was added and incubated at 371C overnight. Chymotrypsin digestions were performed by addition of 25 mM ammonium bicarbonate containing 25 ng/mL. Chymotrypsin (sequencing grade; Roche Diagnostics) was incubated for 2.5 h at 301C. Peptides were extracted with 35 mL of 20 mM ammonium bicarbonate at 371C for 15 min, followed by 70 mL of 2% formic acid/40% ACN at 371C for 15 min, and dried in a SpeedVac to a final volume of 10 mL.
2.7 nano-LC-ESI-MS/MS analysis For Mn-SOD identification, tryptic and chymotryptic digested peptides were separated by the Ultimate 3000 nano-LC system (Dionex, Amsterdam, Netherlands) and analyzed using a QSTAR XL (qQ-TOF; Applied Biosystems, Foster City, CA, USA) equipped with a nano electrospray ionization source [27]. For nano-LC-ESI-MS/MS, the digest was loaded onto PepMap 100 C18 precolumn (300 mm id, 5 mm long cartridge, from Dionex) from 0 to 30 min and then separated by PepMap 100 C18 analytic column (75 mm id 150 mm long cartridge, from Dionex) using a linear gradient of 4% B (Solvent A: 0.1% formic acid; Solvent B: 80% ACN/0.08% formic acid) to 60% from 0 to 30 min, 90% B constant from 30 min 35%, 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 www.electrophoresis-journal.com
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350–1600) followed by MS/MS spectra on the three most abundant peptide ions in the full MS scan. The derived MS data sets were converted to MASCOT generic format flat files by macot.dll 1.6b21 (Matrix Science, Boston, MA, USA) script supplied with AnalystQSs 1.1 software (Applied Biosystems) and searched against in house-licensed MSDB 20051115 and UniProtKB databases. For protein identification oxidation of methionine and deamidation of Gln and Asn were set as variable modifications, carbamidomethylation of cysteine residues as fixed modification.
2.8 Analysis by nano-LC-ESI-(CID/ETD)-MS/MS (high-capacity ion trap) In-gel digestion and sample preparation for high-capacity ion trap (HCT) analysis was performed as described previously [28]. Gel plugs were washed with 10 mM ammonium bicarbonate and 50% ACN in 10 mM ammonium bicarbonate repeatedly. Addition of 100% ACN resulted in gel shrinking and the shrunk gel plugs were then SpeedVac dried in a SpeedVac Concentrator 5301 (Eppendorf, Germany). The dried gel pieces were reswollen and digested with 40 ng/mL trypsin (Promega) in 10 mM ammonium bicarbonate and incubated overnight at 371C. Peptides were extracted with 35 mL of 20 mM ammonium bicarbonate at 371C for 15 min, followed by 70 mL of 2% formic acid/40% ACN at 371C for 15 min, and volume was reduced in a SpeedVac to a final volume of 20 mL. A total of 15 mL of extracted peptides was analyzed by HCT. The HPLC used was a biocompatible Ultimate 3000 system (Dionex, Sunnyvale, CA, USA) equipped with a PepMap100 C-18 trap column (300 mm 5 mm) and PepMap100 C-18 analytic column (75 mm 150 mm). The gradient was (A 5 0.1% formic acid in water, B 5 0.08% formic acid in ACN) 430% B from 0 to 105 min, 80% B from 105 to 110 min, 4% B from 110 to 125 min. The flow rate was 300 nL/min from 0 to 12 min, 75 nL/min from 12 to 105 min, and 300 nL/min from 105 to 125 min. A HCT ultra PTM discover system (Bruker Daltonics) was used to record peptide spectra over the mass range of m/z 3501500, and MS/MS spectra in information-dependent data acquisition over the mass range of m/z 1002800. Repeatedly, MS spectra were recorded followed by three data-dependent CID MS/ MS spectra and three ETD MS/MS spectra generated from three highest intensity precursor ions. An active exclusion of 0.4 min after two spectra was used to detect low-abundance peptides. The voltage between ion spray tip and spray shield was set to 1100 V. Drying nitrogen gas was heated to 1701C and the flow rate was 10 L/min. The collision energy was set automatically according to the mass and charge state of the peptides chosen for fragmentation. Multiple charged peptides were chosen for MS/MS experiments due to their good fragmentation characteristics. MS/MS spectra were interpreted and peak lists were generated by DataAnalysis 3.4 (Bruker Daltonics). Searches were completed by using the MASCOT 2.2.04 (Matrix Science) against latest & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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UniProtKB database for protein identification. Searching parameters were set as follows: enzyme selected as trypsin with two maximum missing cleavage sites, species limited to human, a mass tolerance of 0.25 Da for peptide tolerance, 0.25 Da for MS/MS tolerance, fixed modification of carbamidomethyl (C) and variable modification of methionine oxidation. Positive protein identifications were based on a significant MOWSE score. After protein identification, an error-tolerant search was completed to detect unspecific cleavage and unassigned modifications. Protein identification and modification information returned from MASCOT were manually inspected and filtered to obtain confirmed protein identification and modification lists of CID MS/MS and ETD MS/MS.
2.9 PTMs identification using MASCOT, Phenyx and Modiro 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 UniProtKB database. For PTMs identification, oxidation of histidine, nitration of tyrosine and oxidation of Trp were set as variable modifications, carbamidomethylation of cysteine residues as fixed modification. Top scoring candidate peptides were manually validated with MASCOT results. For Modiro PTMs identification, CID and ETD spectra were converted into mfg file and submitted for PTMs search. For PTM identification, basic search options were MS tolerance of 0.2 Da and MS/ MS tolerance 0.2 Da, oxidation of methionine and carbamidomethylation of cysteine residues was selected. For advanced PTM-Explorer search strategies, search for unknown mass shift, search for amino acid substitution and calculate significance, was selected. Scores for peptide modification greater than 80 were considered significant.
3 Results 3.1 Protein sequence analysis By protein profiling of the human medulloblastoma cell line, two protein spots were identified as Mn-SOD with identical molecular weights and same pI (Fig. 1). To determine sequence identity and PTM, an MS approach was carried out. Two protein spots from 2-DE gels were picked and digested individually with trypsin, Asp-N and chymotrypsin analyzed with MALDI-TOF/TOF, nano-LCESI-MS/MS and ion trap. MALDI-TOF/TOF and nano-LCESI-MS/MS analyses confirmed the absence of the transit peptide of Mn-SOD (amino acid numbers 1–24 in both spot 1 and spot 2 protein spots). Protonated molecular ions of tryptic peptide ions observed at m/z 3352.70 (25-KHSLPDLPYDYGALEPHINAQIMQLHHSK-53) were www.electrophoresis-journal.com
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75
50
37
25
20
4
5
5.5
6
Spot 1
Spot 2
6.8
8
9 pI
Figure 1. Proteins from DAOY cells were subjected to isoelectric focusing on an 18 cm immobilized pH 3–10 gradient strip and then separated by SDS-PAGE. A 2-DE gel image of a Coomassie Blue-stained gel is shown. Mn-SOD protein spots (encircled) are identified and characterized by MALDI-TOF/TOF, nano-LC-ESIMS/MS and nano-LC-ESI-(CID/ETD)-MS/MS.
identified using MALDI-TOF/TOF analysis corresponding to a reported transit peptide cleavage (amino acid numbers 1–24) in both spots (Table 1 and 2). Chymotryptic digests of Mn-SOD protein spots analyzed with nano-LC-ESI-MS/MS identified the sequence 25-KHSLPDLPY-33 observed at m/z 535.34 (doubly charged ion). Data obtained from MS indicated that 97% of total sequence coverage was obtained by MS/MS analysis when excluding the transit peptide in both Mn-SOD protein spots. Enzymatic digestion with different proteolytic enzymes could not give the full sequence information: The C-terminal peptide 194–198 could not be identified by MALDI-TOF/TOF, nano-LC-ESIMS/MS and ion trap. To resolve this uncertainty, we decided to carry out in-gel acetylation of protein lysine residue using N-acetoxy-(1H3) succinimide and the labeled protein spots were digested with trypsin and analyzed with nano-LC-ESIMS/MS. MS data indicated that a doubly charged ion observed at m/z 450.7013 was assigned to the C-terminus peptide 193-YM(Ox)ACK(Ac)K(Ac)-198 in its methionineoxidized state; both Lys-197 and 198 were modified with acetylation (Table 1 and 2). Herein we demonstrate the complete protein sequence analysis of Mn-SOD protein spots from the medulloblastoma cell line. Based on sequencing cDNA isolated from a human colon carcinoma, Church et al., published a sequence containing asparagine instead of threonine at amino acid number 65 (unprocessed Mn-SOD precursor) [29]. Tryptic digested peptides from spots 1 and 2 analyzed by MALDI& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
TOF/TOF, nano-LC-ESI-MS/MS and ion trap have confirmed the presence of threonine. MS/MS analysis of the tryptic digested peptide of spots 1 and 2 observed at m/z 2542.24 and a subsequent MASCOT search showed that it perfectly matched to Mn-SOD protein (54HHAAYVNNLNVTEEKYQEALAK-89; UniProtKB accession number P04179) with a significant high score. Moreover, nano-LC-ESI-MS/MS and ion trap analysis of a tryptic digested peptide observed at m/z 1738.05 (54-HHA AYVNNLNVTEEK-68) in its double and triple charged ions confirmed the presence of thereonine at amino acid number 65 in human Mn-SOD (UniProtKB accession number P04179). Based on cDNA sequencing analysis, St. Clair et al., proposed a sequence containing glutamine at amino acid numbers 66, 112, 133 and 155 (unprocessed Mn-SOD precursor) instead of the corresponding amino acid glutamic acid [29, 30]. Protonated molecular ions observed at m/z 2542.24, 2637.24 and 2291.15, their corresponding sequences 54-HHAAYVNNLNVTEEKYQEALAK-89, 90-FNGGGHI NHSIFWTNLSPNGGGEPK-114 and EKLTAASVGVQGSG WGWLGFNK confirmed the presence of glutamic acid at 66, 112 and 133. The presence of glutamic acid at amino acid number 155 was confirmed observed at ion m/z 774.39 (135-LTAASVGVQGSGWGWLGFNKER-156) in its deamidated form. As to the finding of Church et al. [29], proposing absence of glycine 148 and Trp 149, we confirm the presence of these two amino acids. Heckl [31] published a cDNA sequence of SOD-2 from placenta containing proline and leucine at amino acid numbers 14 and 123 instead of corresponding amino acids alanine and arginine in human SOD (UniProtKB accession number P04179). Based on MS data, the presence of neither alanine nor proline could be confirmed because of the absence of the transit peptide region. The presence of arginine at amino acid number 123, however, was confirmed at observed ion 486.20 (123-RDFGSFDK-130) in medulloblastoma Mn-SOD.
3.2 Oxidative modification of histidine and Trp MS/MS analysis by the ion trap and nano-LC-ESI-MS/MS analysis of tryptic peptides obtained from Mn SOD (spot 2) indicated an observed [M13H]31ion observed at m/z 590.98 that was not observed in spot 1. This finding shows that spot 2 was a further histidine-oxidized form of Mn-SOD, whereas the spot 1 did not show histidine oxidation. Sequencing analysis of triple charged ions observed at m/ z 590.98 was matching the sequence HHAAYVNNLNVTEEK (residues in bold indicate the amino acids that are modified to 2-Oxo-histidine) in Mn SOD protein spot 2. Histidine oxidation of the peptide increases the mass of an ion by 16 Da and both His-30 and His-31 were modified to 2-Oxo-hisdtidine and thus leading to a mass increment of 32 Da (Fig. 2). The close observation MS/MS spectrum revealed the presence of a b2ion at m/z 307.11, which indicates that both, His-30 and His31, were modified with 2-Oxo-histidine. In addition, as www.electrophoresis-journal.com
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Table 1. Identification of Mn-SOD protein (spot 1) by MS analysisa) Peptides
Experimental [M1H]1 ion (m/z)
Calculated [M1H]1 ion (m/z)
Charge state
Enzymeb)
MSc)
1
1068.66 3351.70 1738.05 2542.32 1321.79 821.54 835.43 1423.96 2636.28 1581.98 871.61 885.58 1027.57 877.56 1168.72 1558.97 970.56 814.46 1003.67 1089.65 1617.02 2291.15 2033.93 2324.16 1492.65 1323.774 1506.85 2079.92 1679.78 3195.49 1502.00 1003.65 1742.81 1746.82 1756.83 1758.84 1774.84 1758.98 1790.86 1790.85 1535.90 909.52 899.53
1068.56 3351.68 1737.84 2541.26 1321.65 821.43 835.44 1423.80 2636.25 1581.80 871.50 885.52 1027.60 877.47 1168.59 1558.78 970.45 814.35 1003.42 1089.51 1616.85 2291.17 2034.03 2323.06 1492.65 1322.604 1506.87 2080.02 1679.84 3195.63 1501.84 1003.55 1742.87 1746.86 1756.88 1758.87 1774.86 1758.94 1790.85 1790.85 1535.74 909.42 899.55
21 11 11, 31, 21 21 21 21, 11, 21 11, 31 31 21 21 31 21 21 21 11, 31 21, 21, 31 11 21 11 11 11 11 31 11, 11, 11, 31 31 31, 11 11 11 21 21 21
C T T T C T T T T A T T T C C C T T T T C TA T T A T A A A A C T T T T T T
E M ME ME E EI E EI ME M ME I E E E E E EI E ME E ME E E M E M M M M E ME ME IM I E MI M M M E E E
KHSLPDLPY9 KHSLPDLPYDYGALEPHINAQIMQLHHSK29 30 HHAAYVNNLNVTEEK44 30 HHAAYVNNLNVTEEKYQEALAK51 35 VNNLNVTEEK44 45 YQEALAK51 45 YQEALAK51 Methyl E 52 GDVTAQIALQPALK65 66 FNGGGHINHSIFWTNLSPNGGGEPK90 79 TNLSPNGGGEPKGELL94 91 GELLEAIK98 91 GELLEAIK98 Methyl E 91 GELLEAIKR98 95 EAIKRDF101 95 EAIKRDFGSF104 95 EAIKRDFGSFDKF107 99 RDFGSFDK105 100 DFGSFDK105 100 DFGSFDK(Ac)F106 100 DFGSFDKFK107 108 KEKLTAASVGVQGSGW123 109 EKLTAASVGVQGSGWGWLGFNK130 111 LTAASVGVQGSGWGWLGFNK130 111 LTAASVGVQGSGWGWLGFNKER132 131 ERGHLQIAACPNQ143 d) 132 GHLQIAACPNQD144 144 DPLQGTTGLIPLLGI158 159 DVWEHAYYLQYKNVRP174 162 EHAYYLQYKNVRP174 162 EHAYYLQYKNVRPDYLKAIWNVINW186 170 KNVRPDYLKAIW181 171 NVRPDYLK178 179 AIWNVINWENVTER192 179 AIWNVINWENVTER192 Trp-4Kynurenin (W) 179 AIWNVINWENVTER192 e) 179 AIWNVINWENVTER192 Trp-4hydroxytryptophan 179 AIWNVINWENVTER192 Trp-4N-formylkynurenine 179 AIWNVINWENVTER192 Trp-43-hydroxykynurenine 179 AIWNVINWENVTER192 Trp-4hydroxy-N-formylkynurenine 179 AIWNVINWENVTER192 Trp-4hydroxytryptophan. 182 NVINWENVTERY193 187 ENVTERY193 193 YM(Ox)ACK(Ac)K(Ac)198 1
21, 31 11
31 21 21, 31
21, 31 11 31
21, 31 21, 31 31
11
T T C C T
I
I I
I I
a) Letters in bold indicate amino acid site modified by oxidation of methionine [Oxi(M)] and Trp [Oxi(W)] or methylation of glutamic acid. b) Key: T 5 trypsin, A 5 Asp-N and C 5 chymotrypsin. c) Key: M, MALDI-TOF/TOF, E, nano-LC-ESI-MS/MS and I, nano-LC-ESI-(CID/ETD)-MS/MS. d) Indicates that sequences were derived from PMF data. e) Indicates identification of unknown by-product Trp found at Trp-186.
compared with non-oxidized peptide fragments, y2–y9-singly charged fragmented masses were unchanged. The presence of a double charged mass of y11 (m/z 661.8) and y13 (m/z 732.8) proposes the 32 Da mass increments located at y14 and y15 ion fragments. In turn, the presence of an ion at m/z 800.87 corresponds to a loss of ammonia from y14, indicating that
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His 31 was modified by a mass shift of 16 Da. These results along with the presence of a doubly charged y15 ion at m/z 885.92 and the presence of an ion at m/z 876.91, corresponding to water loss from doubly charged y15-ion demonstrated that His-30 and His 31 were modified with 2-Oxohistidine.
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Table 2. Identification of Mn-SOD protein (spot 2) by MS analysisa) Peptides
Experimental [M1H]1 ion (m/z)
Calculated [M1H]1 ion (m/z)
Charge state
Enzymeb)
MSc)
1
1068.66 3351.70 1738.05 2542.32 1321.79 821.54 835.43 1423.96 2636.28 1581.98 871.61 885.58 1027.57 877.56 1168.72 1558.97 970.56 814.46 1089.65 1617.02 2291.15 2033.93 2005.17 1081.62 2416.26 1492.65 1506.85 2079.92 1679.78 3195.49 1502.00 1003.65 1533.77 1976.94 1742.81 1746.84 1756.86 1758.85 1774.83 1758.96 1790.86 1790.86 1535.90 909.52 899.53
1068.56 3351.68 1737.84 2541.26 1321.65 821.43 835.44 1423.80 2636.25 1581.80 871.50 885.52 1027.60 877.47 1168.59 1558.78 970.45 814.35 1089.51 1616.85 2291.17 2034.03 2004.98 1081.53 2416.36 1492.65 1506.87 2080.02 1679.84 3195.63 1501.84 1003.55 1533.80 1977.00 1742.87 1746.86 1756.87 1758.86 1774.82 1758.94 1790.85 1790.85 1535.74 909.42 899.39
21 11 11, 31, 21 21 21 21, 11, 21 11, 31 31 21 21 31 21 21 11, 31 21, 21, 21 21 21 11 11 11 11 11 31 11, 11 11 11, 11, 31 31 31, 11 11 11 21 21 21
C T T A C T T T T A T T T C C C T T T C TA T TU U U A A A A A C T A A T T T T T
E M ME ME E EI E EI ME M ME I E E E E E EI ME E ME E T I I M M M M M E ME M M ME MI I E MI M M M E E E
KHSLPDLPY9 HSLPDLPYDYGALEPHINAQIMQLHHSK29 30 HHAAYVNNLNVTEEK44 30 HHAAYVNNLNVTEEKYQEALAK51 35 VNNLNVTEEK44 45 YQEALAK51 45 YQEALAK51 Methyl E 52 GDVTAQIALQPALK65 66 FNGGGHINHSIFWTNLSPNGGGEPK90 79 TNLSPNGGGEPKGELL94 91 GELLEAIK98 91 GELLEAIK98 Methyl E 91 GELLEAIKR98 95 EAIKRDF101 95 EAIKRDFGSF104 95 EAIKRDFGSFDKF107 99 RDFGSFDK105 100 DFGSFDK105 100 DFGSFDKFK107 108 KEKLTAASVGVQGSGW123 109 EKLTAASVGVQGSGWGWLGFNK130 111 LTAASVGVQGSGWGWLGFNK130 114 SVGVQGSGWGWLGFNK (Ac)ER132 d) 133 GHLQIAACPN142 133 GHLQIAACPNQDPLQGTTGLIPL155 131 ERGHLQIAACPNQ143 e) 144 DPLQGTTGLIPLLGI158 159 DVWEHAYYLQYKNVRP174 162 EHAYYLQYKNVRP174 162 EHAYYLQYKNVRPDYLKAIWNVINW186 e) 170 KNVRPDYLKAIW181 171 NVRPDYLK178 175 DYLKAIWNVINW186 175 DYLKAIWNVINWENVT190, 179 AIWNVINWENVTER192 179 AIWNVINWENVTER192 Trp-4Kynurenin (W) 179 AIWNVINWENVTER192 f) 179 AIWNVINWENVTER192 Trp-4Hydroxytryptophan 179 AIWNVINWENVTER192 Trp-4N-formylkynurenine 179 AIWNVINWENVTER192 Trp-43-hydroxykynurenine 179 AIWNVINWENVTER192 Trp-4hydroxy-N-formylkynurenine 179 AIWNVINWENVTER192 Trp-4hydroxytryptophan. 182 NVINWENVTERY193 187 ENVTERY193 193 YMACK(Ac)K(Ac)198 1
21, 31 11
31 21 21, 31
21, 31 11 31
21, 31
21, 31 31
11
T T C C T
I
I I
I
I
a) Ac indicates chemically acetylated lysine residue. Letters in bold indicate amino acid site modified by oxidation of methionine [Oxi(M)] and Trp [Oxi(W)] or methylation of glutamic acid. b) Key: T, Trypsin, A, Asp-N and C, chymotrypsin; TU indicates that a peptide is derived from unspecific cleavage. c) Key: M, MALDI-TOF/TOF, E, nano-LC-ESI-MS/MS and I, nano-LC-ESI-(CID/ETD)-MS/MS. d) Indicates that peptide was identified with in-gel chemical labelling method. e) Indicates the sequences that were derived from PMF data. f) Indicates identification of unknown by-product Trp found at Trp-186.
The ion trap MS/MS spectrum of a triple-charged peptide ion observed at m/z 589.636 shows that the peptide HHAAYVNNLNVTEEK mass increased to 28 Da and & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
the spectrum indicated that the mass of His-30 and His-31 was increased to 28 Da (Fig. 3). As compared with the unmodified peptide fragments, singly charged y2–y9 www.electrophoresis-journal.com
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Figure 2. Identification of the 2-Oxo-histidine sites in MnSOD by nano-LC-ESI-(CID/ ETD)-MS/MS analysis. The MS/MS spectrum of the tryptic peptide HHAAYVNNLNVTEEK (spot 2) shows that His30 and His-31 were modified with 2-oxo-histidine as indicated by the presence of a b2-ion at m/z 307.11 (mass shift 132 Da; in the spectrum labeled with HH132). Details are provided in Section 3.
Figure 3. Identification of a proposed histidine oxidation intermediate by nano-LC-ESI(CID/ETD) MS/MS analysis. The MS/MS spectrum of the tryptic peptide HHAAYVNNLNVTEEK (spot 2) shows that His-30 and His-31 were modified with a proposed histidine oxidation intermediate (His114) as indicated by the presence of a b2ion at m/z 303.05 (mass shift 1 14 Da; in the spectrum labeled with HH114).
and doubly charged y5–y11 were unchanged, thus localizing the 24 Da mass increments between y12–y15. The observation of an ion at m/z 689.35 corresponding to loss of ammonia from the doubly charged y12 ion, ruled out the possibility that y12 was modified. The mass spectra clearly showed the presence of b2 ion at m/z 303.06, which corresponds to b1 and b2 ions mass increase to 28 Da. In addition, the mass of singly charged b4–b9 ions and doubly charged b3–b14 ion series is shifted to & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
28 Da as well, indicating that the 24 Da mass increment was assigned to b1 and b2 ions. MS data failed to show whether b1 and b2 ions were modified by 14 Da each or 28 Da mass increases in either one of the histidine residues. Most probably the possible unknown mass shift of 28 Da may reflect possible intermediates of histidine oxidation at His-31 and His 32. Trp oxidation products were observed in both forms of Mn-SOD protein. Tryptic peptides analyzed with MALDIwww.electrophoresis-journal.com
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Unmodified AIWNVINWENVTER
hyroxytryptophan
N-formylkynurenine hydroxy-N-formylkynurenine
kynurenine (+4Da) 3-hydroxykynurenine Unknown by-product
1757.8
Figure 4. Trp oxidation products as revealed by MALDI-TOF analysis. Trp derivatives obtained from m/z 1743. 9 peak that corresponds to the peptide containing the unmodified Trp residue, an ion at m/z 1747.88 corresponds to kynurenine, an ion at m/z 1757.82 could correspond to an unknown by-product found in all Trp oxidized patterns. An ion at m/z 1759.89 may represent hydroxytryptophan, an ion at m/z 1763.87 may represent 3-hydroxykynurenine, an ion at m/z 1775.88 to the N-formylkynurenine and another ion at m/z 1791.87 may represent hydroxy-Nformylkynurenine.
Figure 5. MALDI-TOF/TOF analysis of N-formylkynurenine. MS/MS spectrum of the ion at m/z 1775. 86 from tryptic digests of Mn-SOD (spot 1) showing that Trp-186 was modified to N-formylkynurenine. The asterisk indicates that the mass of y6 and y7 increased to 32 Da, representing N-formylkynurenine.
TOF/TOF revealed the presence of Trp oxidation products. Figures show MALDI-TOF (MS) analysis of tryptic peptide of matching sequence 179-AIWNVINWENVTER-192 and various oxidation products that were observed in both MnSOD protein spot 8 (Fig. 4). The Trp 186 residue was oxidized to products such as kynurenine (14 Da), hydroxytryptophan (16 Da), 3-hydroxykynurenine (120 Da), N-formylkynurenine (132 Da) and hydroxy-N-formylk-
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ynurenine (148 Da). MALDI-TOF data indicate that an observed ion at m/z 1747.88 corresponds to kynurenine (Supporting Information Fig. 1), an observed ion at m/z 1757.82 could correspond to an unknown by-product found in all Trp-oxidized patterns (Supporting Information Fig. 2). An observed ion at m/z 1759.89 may represent hydroxytryptophan (Supporting Information Fig. 3), an ion at m/z 1763.87 to 3-hydroxykynurenine, another observed ion at m/
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z 1775.88 to the N-formylkynurenine, and an observed ion at m/z 1791.87 may represent hydroxy-N-formylkynurenine. The peptide179-AIWNVINWENVTER-192, however, contains two Trp residues; hence, site-specific localization of oxidized Trp residue had to be respected: MS/MS analysis of Mn-SOD tryptic peptides using MALDI-TOF/TOF, nanoLC-ESI-MS/MS and ion trap was carried out. Data derived from MS were subsequently searched with MASCOT to identify modifications and in addition were completed with Phenyx and Modiro PTM softwares to validate the modifications. Data from MS clearly indicated that Trp-186 and not Trp-181 undergoes oxidation. Figure 5 shows the MS/MS spectrum of a singlecharged tryptic peptide modified with N-formylkynurenine.
4 Discussion Several reports on aberrant expression of Mn-SOD and oxidative stress in cancer cells [18, 32, 33] have been published and, more specifically, a role for Mn-SOD in tumor suppression has been proposed [34–36]. These important findings warranted chemical studies on this key player in tumor biology. And indeed, Mn-SOD(s) in tumor cells have not been fully characterized neither complete protein sequences, nor have PTMs, pivotal for function of a protein, been sufficiently addressed. Carrying out protein profiling in medulloblastoma cells [19], we observed two spots on 2-DE. As individual Mn-SODs were reported in different tumor cells, we may have expected the presence of a splice variant. Complete sequencing of both spots with apparently comparable molecular weights, however, revealed 100% of identity. This fact suggests that the shift in pI was induced by a PTM. Although other reports on tumor cells have shown two spots for Mn-SOD, no characterization was provided, i.e. only partial protein sequences were published. Complete sequencing of proteins was accomplished by the combination of multi-enzyme digestion, an in-gel labeling method and different MS principles. Identification of PTMs of spot 2 showed oxidized His-30 and His-31 but this modification was not observed in spot 1. Several lines of evidence indicate mechanisms of oxidative damage and metal-catalysed oxidation [37, 38]. Histidine is one of the amino acids, most vulnerable to oxidative reactions. In the current study we detected for the first time the oxidation of neighboring amino acids His-30 and His-31. Both amino acids were modified to 2-oxo-histidine, and an intermediate His114 published previously [39]. Side-chain imidazole of His-30 forms two hydrogen bonds in Mn-SOD, one with the side-chain hydroxyl of Tyr34 through an intervening water molecule and a second with the side-chain hydroxyl of Tyr-166 from the adjacent subunit in the dimer and side chain partially exposed to water. Histidine oxidation may be leading to disturbed functions including substrate accessibility, catalytic activity and redox chemistry of metal (Mn) binding and there is indirect evidence from site-directed mutagenesis [40, 41]. & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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The mechanism of 2-oxo-histidine formation remains unclear: However, a mechanism of metal-catalyzed oxidation of histidine to 2-oxo-histidine in peptides and proteins has been proposed [42] and Uchida and Kawakishi identified 2-oxo-histidine using HPLC with electrochemical detection in Cu,Zn-SOD in vitro [43, 44]. A MS-based 2-oxo-histidine detection method has been developed to identify site specificity of this modification in protein and peptides [45]. In the current study the identified peptide HHAAYVNNLNVTEEK was modified with a 28 Da mass shift of His-30 and His-31. We propose that the 28 Da mass shift of the peptide (His1 14 for each histidine residue) may represent an intermediate product of His-30 and His-31. Chang et al., proposed a scheme for metal catalyzed photo-oxidation of histidine: one of the several intermediates is histidine containing doublebonded oxygen plus two hydroxyl groups (His150 Da) and the loss of two water molecules from this product give rise to the His114 Da product [39]. Identification of triple-charged peptide ion at m/z 589.636 could be the abovementioned product. Other intermediates of histidine oxidation were not detected, probably because of instability of these products in solution [39]. This work therefore provides more specific information about oxidation histidine in human Mn-SOD and the modification may be possible role in tumor formation that remains to be evaluated in further functional studies. We identified Trp oxidation and observed specific products as kynurenine, hydroxytryptophan, 3-hydroxykynurenine, N-formylkynurenine and hydroxy-Nformylkynurenine in both spots analyzed. This is in agreement with findings from Zhang et al., who showed the presence of kynurenine (of Trp-32) in human Cu,Zn-SOD [46] and Yamakura and Ikeda showed Trp oxidation along with nitrated products in human Cu,Zn-SOD using microLC-ESI-Q-TOF mass analyses and HPLC with a photodiode-array detector [47]. No nitration of Mn-SOD, however, was detected in the present study. The presence of Trp oxidation products in both proteins in contrast to histidine oxidation may well have been due to technical reasons including sample preparation. Although one could get additional information by studying a non-tumor cell line, studies are hampered by the fact that the original cell type from which medulloblastomas develop has not been identified so far. In conclusion, two spots representing Mn-SOD were fully sequenced and histidine oxidation may be responsible for the shifts in pI as revealed by 2-DE. Although the complete sequence is important for further chemical and immunochemical studies as e.g. antibody generation, the oxidative modifications of histidine may be of relevance for function of the Mn-SOD activity in medulloblastoma cells. These results will have to be respected for interpretation of previous reports and future designs of Mn-SOD studies. The authors are highly indebted to Drs. Irene Slavc, Mariella Gruber-Olipitz and Thomas Stroebel for providing gels from a previous common study and to Dr. Wei-Qiang Chen for www.electrophoresis-journal.com
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HCT analysis of the two Mn-SOD spots. They acknowledge the contribution by the Verein zur Durchfu¨hrung der wissenschaftlichen Forschung auf dem Gebiet der Neonatologie und Kinderintensivmedizin ‘‘Unser Kind’’. The authors have declared no conflict of interest.
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