4340 Cristina Di Poto1, 2 Paolo Iadarola1 Roberta Salvini1 Ileana Passadore1, 3 Cristina Cereda2 Mauro Ceroni2, 4, 5 Anna Maria Bardoni1 1
Department of Biochemistry, University of Pavia, Pavia, Italy 2 Laboratory of Experimental Neurobiology, IRCCS, Foundation “C. Mondino” Institute of Neurology, Pavia, Italy 3 Department of Haematological, Pneumological and Cardiovascular Sciences, University of Pavia, Pavia, Italy 4 Department of Neurology, Policlinico of Monza, Monza, Italy 5 Department of Neurosciences, University of Pavia, Pavia, Italy
Received March 2, 2007 Revised May 17, 2007 Accepted July 27, 2007
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Research Article
Optimizing separation efficiency of 2-DE procedures for visualization of different superoxide dismutase forms in a cellular model of amyotrophic lateral sclerosis Neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease (PD) have been associated with increased production of reactive oxygen species. In AD and PD patients, superoxide dismutase (SOD1) was also indicated as a major target of oxidative damage. In particular, in brain tissue of these patients, different SOD1 isoforms have been identified, although their functional role still remains to be elucidated. In the light of the possibility that different SOD1 entities could be expressed also in other neurodegenerative disorders, as a sort of unifying event with AD and PD, we have investigated amyotrophic lateral sclerosis (ALS) using human neuroblastoma SH-SY5Y cells with mutated SOD1 gene H46R as cellular model. 2-DE using a narrow-range IPG 4–7 strips in the first dimension and linear 15% SDS-PAGE in the second allowed to separate different SOD1 spots. MALDI-TOF MS and CapLC-MS/MS have been used for their complete identification. This is the first report in which the presence of SOD1 (iso)forms in a cellular model of ALS has been evidenced. Keywords: 2-DE / MALDI-MS / Neurodegenerative disorders / Superoxide dismutase DOI 10.1002/elps.200700162
Introduction
A common characteristic of neurodegenerative and psychiatric diseases is that their molecular bases are unknown and individual biomarkers are often not reliable. This is the case of amyotrophic lateral sclerosis (ALS), Alzheimer disease (AD), and Parkinson disease (PD), chronic disorders caused by a combination of events that impair normal neuronal function and characterized by the accumulation of insoluble proteinaceous deposits [1–3]. Unifying events in these slowly progressive neurodegenerative disorders include accumulation of aberrant or misfolded proteins, protofibril formation, ubiquitin–proteasome system dysfunction, mitochondrial injury, synaptic failure, altered metal homeostasis, and oxidative and nitrosative stress [1, 3]. Both AD and PD have
Correspondence: Professor Paolo Iadarola, Department of Biochemistry, University of Pavia, Via Taramelli 3/B, I-27100 Pavia, Italy E-mail:
[email protected] Fax: 139-0382-423108 Abbreviations: AD, Alzheimer disease; ALS, amyotrophic lateral sclerosis; DTE, dithioerythritol; FALS, familial ALS; NL, nonlinear; PD, Parkinson disease; SOD1, superoxide dismutase
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been associated with increased production of reactive oxygen species, which could result either from genetic predisposition and/or environmental factors such as exposure to pesticides [1, 4]. Markers of oxidative damage have also been identified in the motor cortex and the spinal cord of patients with familial ALS (FALS) and sporadic ALS (SALS) [5–7]. In an effort to obtain a better understanding of the pathogenic mechanisms in these diseases, specific proteins that are targets of oxidative stress have been largely investigated [1, 3, 6]. As a result of these studies, ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) was identified as the causative genetic defect for certain familial forms of PD [8, 9] and Cu,Zn-superoxide dismutase (SOD1), an antioxidant enzyme that plays an essential role in the cellular defense against harmful superoxide radicals, was indicated as a major target of oxidative damage in AD and PD [1]. In particular, a total of four SOD1 isoforms differing in their pI have been identified in brain of PD and AD subjects and a series of mutations in the SOD1 gene encoding for the SOD1 enzyme have been clearly evidenced in individuals with familial ALS [10, 11]. Among these, of particular interest appeared the H46R mutation which involves one of the residues that binds catalytic copper at the active site [12] and originates a SOD with a dramatically reduced activity [11]. Thus, in the light of the possibility that SOD1-modified www.electrophoresis-journal.com
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forms could be present not only in brain but also in other cellular compartments and for neurodegenerative disorders other than PD and AD, we have probed for these entities in ALS using human neuroblastoma SH-SY5Y cells, transfected with mutated SOD1 gene H46R, as cellular model. Given that proteomics technologies have proved to be an ideal research platform for monitoring changes in the expression level of complex protein mixtures, 2-DE profiling combined with Western blotting and MS were the methods of choice for this study. Despite their well-recognized robustness and efficiency, the “conventional” procedures which employed broad-range (pH 3–10) nonlinear (NL) IPG strips for IEF and a 9–16% T for SDS-PAGE in the second dimension, did not show the power to resolve the spots of interest present in our mixture and made assignment of specific protein identity unreliable. We present here an accurate description of the analytical protocol developed for obtaining their separation and identification. The data shown in this study demonstrate for the first time that different SOD1 entities are expressed also in this cell model of ALS, although a great deal of research is still needed to obtain their structural characterization and to establish whether these forms may represent a sort of unifying event between this disorder and PD or AD. Our investigation confirms that proteomics is an exquisite tool for identifying target proteins that may be involved at important time points in human (neurodegenerative) disorders.
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Materials and methods
2.1 Reagents Cell culture media and reagents, protease inhibitor cocktail, 1,4-dithioerythritol (DTE), CHAPS, glycerol, iodoacetamide, agarose, HEPES, KOH, KCl, MgCl2, sucrose, TCA, urea, SDS, Tris, PBS, were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bicinchoninic acid (BCA) was purchased from Pierce (Rockford, IL, USA). Carrier ampholytes and IPG gel strips were from GE Healthcare (Uppsala, Sweden). Antibodies for Cu,Zn-SOD1 and polyclonal anti-Op18 for stathmin/oncoprotein 18 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2.2 Cell lines and cytosol preparation Human neuroblastoma cells SH-SY5Y (SH) were maintained in Dulbecco’s modified Eagle/F12 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, 10 mg/mL streptomycin, at 377C in a atmosphere humidified with 5% CO2. Monoclonal cell line expressing FALS-SOD1 H46R was obtained as previously described [13, 14] by transfection with expression plasmid pRc/CMV (Invitrogen, Carlsbad, CA, USA) containing cDNAs coding for the SOD1 protein. Cytosol from SH-SY5Y © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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cells was prepared using a modified version of method of Yang et al. [15]. Cells were centrifuged at 6006g for 5 min to obtain pellets that were washed twice with ice-cold PBS and resuspended in extraction buffer (20 mM HEPES/KOH pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTE, 2 M sucrose, protease inhibitor cocktail). Cells were homogenized with 30 passes of a micropestle. Homogenates were centrifuged at 8506g for 6 min at 47C originating a postnuclear S1 supernatant that was harvested. The pellet was resuspended in extraction buffer and homogenized as above. The centrifugation step was repeated: the two S1 supernatants were pooled and centrifuged at 13 0006g for 10 min at 47C originating a S2 supernatant whose protein concentration was determined using the BCA assay. Ice-cold TCA (final concentration 12.5%) was added to precipitate proteins. Precipitates were centrifuged at 14 5006g for 30 min and the pellet was washed twice with 1 mL of acetone. Precipitates were finally resuspended in electrophoretic buffer and subjected to 2-DE as indicated in the following paragraph. 2.3 2-DE The IPG gel strips with a NL gradient range of pH 3–10 and a linear gradient of pH 4–7 (length 18 cm in both cases) from GE Healthcare were rehydrated in a swelling solution (350 mL of a buffer containing 8 M urea, 4% w/v CHAPS, 65 mM DTE, 0.8% v/v carrier ampholytes, and 5 mL of bromophenol blue), containing 0.8–2 mg of cytosolic proteins, for 8 h using a low voltage of 30 V at 167C. Each step of IEF was carried out by using the same voltage regime both in case of NL IPG 3–10 or linear IPG 4–7 strips, according to a program driven by the Ettan IPGphor system (1 h at 120 V; 30 min at 300 V; linear ramping from 300 to 3500 V in 3 h; 10 min at 5000 V; and then 7950 V to reach at total of 62 KV/ h). The focused IPG strips were incubated for 12 min at room temperature in a first equilibration buffer containing 6 M urea, 2% w/v SDS, 50 mM Tris (pH 6.8), glycerol 30%, and for 5 min in second equilibration buffer containing 2.5% w/v iodoacetamide and 2% w/v DTE [16]. The strips were hold in place with 0.4% low-melting temperature agarose and loaded onto 20618 cm 9–16% or constant 15% SDSpolyacrylamide gels. Electrophoresis was carried out at a constant current of 40 mA in a PROTEAN® II xi 2-D Cell (BioRad, Richmond, CA, USA) apparatus. The 2-D gels were stained with “Blue silver” according to the manufacturer’s instructions [17]. Data were analyzed using the PDQuest Version 7.2 software (BioRad). 2.4 Western blotting The separated proteins were transferred onto NC membranes by using a Trans-Blot® Electrophoretic Transfer Cell (BioRad, Hercules, CA, USA) and applying a voltage of 100 V for 2 h. Individual membranes were incubated with monoclonal and polyclonal anti-SOD1 specific antibodies for www.electrophoresis-journal.com
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Cu,Zn-SOD1 and polyclonal anti-Op18 for stathmin/oncoprotein 18. Membranes were washed and proteins visualized using an ECL kit. 2.5 In situ digestion and MALDI-MS analysis When 2-D runs were performed for preparative purposes, selected protein spots were excised from the gel and washed in 50 mM ammonium bicarbonate (pH 8.0) and 50% ACN until complete destaining. The proteins were dried under vacuum, resuspended in 50 mM ammonium bicarbonate (pH 8.0), and digested with sequencing grade trypsin (5 mg/ mL, Promega, Madison, WI, USA) at 377C. After incubation overnight, peptides were extracted sequentially three times with 50% ACN, 5% TFA in water. Each extraction involved 10 min of stirring followed by centrifugation and removal of the supernatant. The original supernatant and those obtained from sequential extractions were combined and dried. The peptide mixture was then solubilized with 0.5% TFA for MS analysis. MALDI-TOF analysis of the trypsin digest from the 2-D gel was performed using a Voyager DEPro Applied Biosystem mass spectrometer (Foster City, CA, USA) equipped with a pulsed N2 laser operating at 337 nm. Positive ions spectra were acquired in reflector mode over a m/z range of 500–5000. The analysis was performed by spotting 1 mL of sample mixed with an equal volume of the matrix solution (a-cyano-4-hydroxycinnamic acid 10 mg/mL in 1:1 ACN/water containing 0.1% TFA) onto the target plate. External mass calibration was performed using the peptide mass standard kit provided by manufacturer. Database searches were performed against the NCBI nonredundant database using MASCOT (Matrix Science; www.matrixscience.com) search engine. MASCOT scores greater than 65 were significant (p ,0.05) and were subsequently blasted against the Swiss-Prot database (http:// www.expasy.ch/sprot). 2.6 LC-MS/MS analysis When the identity of proteins could not be established by PMF, the peptide mixtures were further analyzed by LC-MS/ MS using a CapLC capillary chromatography system (Waters, Massachusetts, USA) coupled online with a Q-TOF Ultima hybrid mass spectrometer (Micromass, Toronto, Canada) equipped with a Z-spray source. The peptide mixture (10 mL) was first loaded onto a RP trap column (Waters), using 0.2% formic acid as eluent, at a flow rate of 10 mL/min. The sample was then transferred to a C18 RP capillary column (75 mm620 mm) at a flow rate of 280 nL/min and fractionated using a linear gradient from 7% eluent A (0.2% formic acid in 5% ACN) to 60% eluent B (0.2% formic acid in 95% ACN) in 50 min. The mass spectrometer was set up in a datadependent MS/MS mode to alternatively acquire a full scan (m/z acquisition range from 400 to 1600 Da/e) and a tandem mass spectrum (m/z acquisition range from 100 to 2000 Da/ e). The three most intense peaks in any full scan were © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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selected as precursor ions and fragmented by collision energy. For the peptide sequence searching, the mass spectra were processed and analyzed using the MASCOT MS/MS ion search program with the Swiss-Prot 49.1 database. The mass accuracy was within 50 ppm for the peptide mass tolerance and within 0.25 Da for fragment mass tolerance. Protein identification was repeated at least once using matching spots from different gels. 2.7 N-Terminal sequence analysis Sequence analyses were performed on the HP G1005A (Hewlett-Packard, Palo Alto, CA, USA) protein sequencing system using the routine 3.0 chemistry, according to the manufacturer’s protocol. 2.8 Reproducibility of the study All gels were run in triplicate within the same day and maps presented in this work are the best-representative 2-D gels that showed spots constantly present.
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Results and discussion
3.1 Separation of cytosolic proteins on NL IPG 3–10 strips and immunoblotting Total cytosolic proteins (750 and 800 mg) from SH-SY5Y human neuroblastoma cells (used as model of reference), and from the same cells stably transfected with H46R human SOD1, were separated “in parallel” for the first dimension on 18 cm, pH 3–10 NL IPG gel strips. The focused IPG strips were then loaded onto 20618 cm, 9–16% SDS-polyacrylamide slabs for the second-dimensional run. Detailed maps of proteins exhibited by these samples after staining with colloidal Coomassie are shown in Fig. 1, panels a and b, respectively. Given the aim of our study, from among a total of 176 and 289 spots observed in protein patterns a and b, we focused our interest only on Coomassie-reactive proteins displaying reactivity against anti-SOD1 antibody. Thus, to determine the exact location of SOD1 spots on the gels, proteins separated by 2-DE were electroblotted onto NC membranes coated with a rabbit polyclonal anti-SOD1 antibody. The good matching between the immunosignals shown in panels c and d of Fig. 1 and spots I–III or I–IV in the region of the maps marked by dotted lines in panels a and b, allowed to assign tentatively SOD1 identity to these latter. To achieve the unambiguous identification of these proteins, reactive spots I–IV in panel b were excised, washed to a complete destaining and submitted to in-gel trypsin digestion following the procedure described in Section 2. PMF of spots and database search with the peptide mass spectra obtained allowed to produce the data summarized in Table 1. Spots III and IV were easily identified as human Cu,Zn-SOD1 (SwissProt accession number P00441 and NCBI accession number www.electrophoresis-journal.com
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Figure 1. 2-DE gel images of total cytosol proteins from SH-SY5Y human neuroblastoma cells (“control”, panel a) and from the same cells stably trasfected with H46R human SOD1 (panel b). Separation was performed on 18 cm long IPG strips with a wide-range NL pH gradient 3–10, followed by SDS-PAGE in a 9–16% linear gradient gel. Protein detection was by Coomassie staining. Panels c and d: blot of the windows defined in panels a and b, respectively. Arrows indicate spots displaying SOD1 activity (annotated I–III in panel c and I–IV in panel d) that were located by matching the two images and that were further identified by MALDI-TOF MS. Proteins annotated V and VI will be discussed below (see caption of Fig. 3).
Table 1. List of cytosolic proteins identified in spots indicated I–IV in Fig. 1, panel b
Spot
Protein
NCBI
Observed pI
Theoretical pI
Observed mol. mass (kDa)
Theoretical Sequence mol. mass coverage (kDa) (%)
MOWSE score
I II III IV
Stathmin1/oncoprotein 18 Stathmin1/oncoprotein 18 SOD1 Variant SOD1
gi/61363614 gi/61363614 gi/38489880 gi/31615965
5.50 5.58 5.81 5.92
5.76 5.76 5.70 5.70
19.75 19.00 19.75 19.75
17.35 17.35 16.05 16.05
69 132 122 119
gi/38489880) and Apo-H46R Familial ALS Mutant Human Cu,Zn-SOD1 (Swiss-Prot accession number P00441 and NCBI accession number gi/31615965), respectively. In contrast, the two spots I and II were identified as stathmin, also referred to as oncoprotein 18 (Swiss-Prot accession number P16949; NCBI accession number gi/61363614). Stathmin is a regulatory phosphoprotein known to play the role of a sec© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
66 80 83 80
ond toxic messenger in the pathogenesis of neurodegeneration in FALS with SOD1 mutations [18]. Although the location of stathmin in the region of the gel mentioned above was in good agreement with its molecular size (Mr around 17.225 Da and pI value around pH 5.89 according to ref. [19]), nevertheless the fact that this protein reacted with antiSOD1 antibody generated ambiguity. Thus, to examine whewww.electrophoresis-journal.com
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ther recognition of stathmin could be ascribed to nonspecific crossreactivity exhibited by the antibody adopted, the blotting was repeated using a mouse monoclonal anti-SOD1 antibody. The results obtained, however, were rather disappointing, the distribution of spots responsive to mAb being identical to that shown in panel c. It means that MS spectra of spots I–IV produced the same results previously reported in Table 1, i.e., confirmed the presence of SOD1 under spots III and IV and of stathmin under spots I an II (data not shown). It was apparent that spots I and II, rather than being homogeneous spots comprising a single polypeptide chain, were envelopes of two or more components. The fact that the putative SOD1 entity was, most likely, the less abundant protein in the bulk of other more abundant proteins, rendered its identification difficult. This hypothesis was strengthened by the insufficient quality of MS sequence data which made it difficult to determine exactly, from this analysis, which might be the proteins of interest within the spots considered. Attempts to enrich the content of putative SOD1 in spots I and II by applying on the gel high-protein load (runs with 1 and 2 mg of protein mixture have been performed) were not successful, the MS sequence data of stathmin being in any case the only good-quality MS data detected in these spots. Thus, given that the current approach was unlikely to precisely decipher the different proteins in spots of interest, it became imperative to answer the question of how to resolve these spot overlaps. 3.2 Separation on a narrow-range IPG 4–7 strips According to the numerous reports so far appeared in the literature [16, 20, 21], the best option to improve resolution
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and thereby minimize spot overlaps seemed to be the use of narrow-range (such as pH 4–7 or 4.5–5.5) IPG strips. In an attempt to maximize the number of detectable proteins in region considered, we performed in the 1-D a series of runs on an 18 cm long pH 4–7 gel. The results shown in Fig. 2, which represents the typical profiling achieved under the experimental conditions mentioned above, were discouraging. Although an advantage could be expected from the improved separation of spots of interest, their mass spectra compared well to those previously discussed and reconfirmed the presence of SOD1 under spots III and IV and of stathmin under spots I and II. Despite supplementary runs using 4.5–5.5 pH strips have been performed to circumvent these difficulties, no improvement was observed in the number and position of spots in comparison with profile of Fig. 2 (data not shown). In a further effort to overcome these limitations and to definitively establish or exclude the presence of SOD1 under spots I and II, we worked also on the optimization of gel porosity.
3.3 Combination of narrow-range IPG 4–7 strip and linear 15% SDS-PAGE A combination of a narrow pH range (pH 4–7) for the firstdimensional IEF and of a linear 15% porosity in the second, provided a powerful tool for obtaining a comprehensive inventory of protein spots in region considered (Fig. 3, panel a). By visual inspection of the profiling shown in Fig. 3 (panel a), it was clear that this approach exhibited a performance higher than that of procedures previously applied yielding a
Figure 2. 2-DE gel image of total cytosolic proteins separated on an 18 cm long IPG strip with a narrow-range pH gradient 4–7, followed by SDS-PAGE in a 9– 16% linear gradient gel. Protein detection was by Coomassie staining. Spots annotated I–IV are the same as those previously observed in Fig. 1.
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map that, at least in the region of interest, contained a greater number and more distinct spots. The eight protein spots I– VIII arrowed in Fig. 3, panel a were submitted to MS analysis and all of them, with the exception of spots V and VI, for which the quality of MS signal was insufficient, could be identified. The results of these analyses may be summarized as follows: spots I, II, and VII corresponded to stathmin (Swiss-Prot accession number P16949; NCBI accession number gi/61363614); spots III and IV corresponded to SOD1 (Swiss-Prot accession number P00441; NCBI accession number gi/38489880) and Apo-H46R Familial ALS Mutant Human SOD1, respectively (Swiss-Prot accession number P00441 and NCBI accession number gi/31615965); and spot VIII corresponded to dUTP pyrophosphatase (NCBI accession number gi/4503423). Once again the quality of MS signal for spots V and VI was not sufficient to allow their unambiguous identification.
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To prove definitively our assumption, supplementary experiments based on the determination of N-terminal protein sequence were performed. An aliquot of protein recovered from gel spots V and VI was submitted to automated Edman degradation providing the results shown in the scheme below:
SOD1 Spot V Spot VI
Sequence [22] Sequence Sequence
10
20
30
atkavcvlkg atkavavlkg atkavcvlkg
dgpvqgiinf dgpvqgiinf dgpvqgiinf
eqkesngpvK eqkesngpvK eqkesngpvK
Taken together, all these data are unambiguously consistent with the presence of SOD1/mutant SOD1 entities under spots V and VI of protein pattern considered.
3.4 Identification of protein in spots V and VI by means of LC-MS/MS To improve the quality of MS signal for spots V and VI, separation was replicated under the experimental conditions indicated above loading the gel with 2 mg of total cytosolic proteins from transfected cells. A good matching between spots obtained in this run and those of previous runs allowed to select with accuracy the candidate proteins for MS analyses. Spots of interest were excised from the gel, digested with trypsin, and the resulting peptides analyzed by LC-MS/MS. For the peptide sequence searching, mass spectra were processed and analyzed using the Swiss-Prot 49.1 database. Up to four and six peptides with high-score sequence were matched from spots V and VI, respectively. The sequence coverage of proteins was 60% for spot V and 66% for one of the two entities in spot VI. The results of MS analyses (summarized in Table 2) allowed to identify in spots V and VI possible (iso)forms of SOD1 and mutant H46R SOD1, respectively, that had never been observed before. The latter entity in spot VI was also identified as SOD1 (NCBI: gi/ 223632), although with lower sequence coverage (43%) and MOWSE score (101) than the former. Four peptides (covering the sequence regions: 10–23; 24–36; 116–128; and 144– 153) out of a total of five identified by MS, were the same as those shown in Table 2. The fifth peptide (covering the sequence 10–30) was peculiar of this entity. The findings described above were further validated by blotting the labeled region of Fig. 3, panel a onto a NC membrane coated with anti-SOD1 mAb. As shown in panel b of the same figure, two (namely spots III and IV), out of the four spots responsive to mAb, were the SOD1 entities identified from the beginning of our experimental approach. The other two, namely spots V and VI, were the newly separated SOD1 forms that overlapped spots of stathmin (spots I and II) in former gels but were well resolved in this latter. The dotted “empty” circles on the membrane indicate the former position (previously shown in Figs. 1 and 2) of stathmin plus SOD1 spots. © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. Panel a: 2-DE gel image of total cytosolic proteins separated on an 18 cm long IPG strip with a narrow-range pH gradient 4–7, followed by SDS-PAGE in a constant 15% gel. Protein detection was by Coomassie staining. Selected spots (I–VIII) analyzed by MS or LC-MS/MS are shown in the region marked by dotted lines. Panel b: Blot of the gel region indicated above onto a NC membrane coated with mouse monoclonal anti-SOD1 antibody. Selected spots (III–VI) analyzed by MS or LC-MS/MS corresponded to SOD1. The dotted empty circles on the membrane (annotated I and IV) indicate the former position of stathmin plus SOD1 previously shown in Figs. 1 and 2.
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Table 2. List of tryptic peptides identified, by LC-MS/MS, in spots indicated V and VI in Fig. 3, panel a
Spot
Protein
NCBI
Matched peptides Start–end Sequence (aa) (No. of peptide)
Sequence MOWSE coverage score (%)
Observed pI
Theoretical Observed Theoretical pI mol. mass mol. mass (kDa) (kDa)
V
SOD1
gi/349912
10–23
GDGPVQ GIINFEQK ESNGPV KVWGSIK TLVVHE KADDLGK LACGV IGIAQ
60
116
5.55
5.70
19.75
16.05
GDGPVQ GIINFEQK ESNGPV KVWGSIK GLTEG LHGFR VHEFGDNTAGC TSAGPHFNPLSR TLVVHE KADDLGK LACGV IGIAQ
66
232
5.60
5.70
19.75
16.05
24–36 116–128 144–153 VI
SOD1
gi/31615955
10–23 24–36 37–46 47–69 116–128 144–153
Aim of the present report was to verify the presence of different SOD1 forms in a cellular model of ALS by applying the classical proteomics procedures. We have experienced that a series of drawbacks, including a large difference in abundance between proteins with very similar pI and Mr, prevent the separation and characterization of spots of interest. The limited sensitivity in the detection of less abundant proteins in fact made their identification difficult in the bulk of other more abundant entities. Nevertheless, the proper choice of pH gradient in the first dimension, of gel porosity in the second and of high-resolution mass spectrometric strategies allowed to obtain their successful separation and unambiguous identification.
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Concluding remarks
A combination of immunoblotting, LC-MS/MS, and N-terminal sequence analysis made identification of different forms of SOD1 in human neuroblastoma SH-SY5Y plasmid pRc/CMV unambiguous. Since these newly identified SOD1-containing spots are located in the gel adjacent to one another and to spots of genuine SOD1, they are most likely characterized by subtle post-translational modifications such as charge variations. In this context, it is tempting to speculate that they may be referred to as “SOD1 isoforms” although further analysis is required to elucidate their structural modifications and, possibly, their precise role and © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
function in ALS. To the best of our knowledge, this is the first report in which the presence of different forms of SOD1 in human neuroblastoma cell cultures is demonstrated.
The authors would like to thank Professor Maria Teresa Carrì, Università di Roma “Tor Vergata”, for invaluable support in the construction of transfected SH-SY5Y cell line and Dr. Angela Flagiello, Università di Napoli Federico II, for performing MALDI-MS analyses. Fabio Ferrari, Marco Fumagalli, and Serena Giuliano (Department of Biochemistry, University of Pavia) are also gratefully acknowledged for their technical support.
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