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BBA - Proteins and Proteomics (v.1774, #11)
Identification of the antitumoral drug emodin binding sites in bovine serum albumin by spectroscopic methods
by Paz Sevilla; José M. Rivas; Francisco García-Blanco; José V. García-Ramos; Santiago Sánchez-Cortés (pp. 1359-1369).
Using SERS, fluorescence, circular dichroism and stopped-flow, we have unequivocally characterized the binding sites of emodin in bovine serum albumin. Emodin interacts with protein through two different binding sites corresponding to Sudlow's sites 1 and 2. Site 2, where the binding drug presents, in the cavity, a form between neutral and mono-anionic species slightly displaced to the neutral one, is the primary interaction site, with higher association binding constant, and hence, higher affinity than the other binding site. This interaction changes considerably the α-helical content of the protein and it occurs mainly within the interval [emodin]/[protein]≤2.0. The process involves a fast reaction and the observed rate constant increases when increasing the [emodin]/[protein] ratio. The secondary emodin interaction site corresponds to the Sudlow's site 1, where the drug shows a similar form to that deduced for site 2, but in this case, it is more displaced to mono-anionic species. This interaction does not change the α-helical content of bovine serum albumin, and it occurs mainly for [emodin]/[protein]>2.0 ratios, the process implies a slower reaction than the union process to the site 2, with an observed rate constant that is invariable within the studied interval.
Keywords: Abbreviations; Arg; arginine; BSA; bovine serum albumin; BSA; f; bovine serum albumin fatty acid free; CD; circular dichroism; DMSO; dimethylsulfoxide; His; histidine; HSA; human serum albumin; HSA; f; human serum albumin fatty acid free; Ile; isoleucine; Leu; leucine; Lys; lysine; MRE; mean residue molar ellipticity; Phe; phenylalanine; Ser; serine; SERS; Surface Enhanced Raman Spectroscopy; Trp; tryptophan; Tyr; tyrosineEmodin; Bovine serum albumin; SERS; Fluorescence; Circular dichroism; Fast reactions in solution; Protein–ligand interaction
High affinity binding of Bcl-xL to cytochrome c: Possible relevance for interception of translocated cytochrome c in apoptosis
by M. Yadaiah; P. Nageswara Rao; P. Harish; Abani K. Bhuyan (pp. 1370-1379).
The release of cytochrome c from mitochondria and apoptosis relies on several preferential and selective interactions involving the Bcl-2 family of proteins. There is, however, no direct evidence for the interaction of cytochrome c with these proteins at any stage of apoptosis. To investigate if any pro-survival protein from the Bcl-2 family could intercept cytochrome c after its translocation from mitochondria, the interaction of cytochrome c with bacterially expressed human Bcl-xL was studied at pH 7. In size-exclusion chromatography, purified full-length His6-tagged Bcl-xL migrated as both dimer and monomer, of which the monomeric fractions were used for experiments. Coimmunoprecipitation studies show that cytochrome c interacts with Bcl-xL. The extent of caspase activity in cell lysate elicited by externally added cytochrome c is reduced when a preincubated mixture of Bcl-xL and cytochrome c is used instead. Equilibrium binding monitored by optical absorption of cytochrome c as a function of titrating concentrations of Bcl-xL yields the association constant, Kass=8.4(±4) ×106 M−1 (binding affinity, Kdiss=1/ Kass≈120 nM) which decreases at high ionic strength. The rates for binding of Bcl-xL to cytochrome c, studied by stopped-flow kinetics at pH 7, show that the bimolecular rate constant for binding, kbi=0.24×106 M−1 s−1. Values of the thermodynamic and kinetic parameters for Bcl-xL–cytochrome c interaction are very similar to those known for regulatory protein–protein interactions in apoptosis.
Keywords: Apoptosis; Bcl-2 protein; Bcl-x; L; Cytochrome; c
Hemocyanin conformational changes associated with SDS-induced phenol oxidase activation
by Sharon Baird; Sharon M. Kelly; Nicholas C. Price; Elmar Jaenicke; Christian Meesters; Dorothea Nillius; Heinz Decker; Jacqueline Nairn (pp. 1380-1394).
The enzymatic activity of phenoloxidase is assayed routinely in the presence of SDS. Similar assay conditions elicit phenoloxidase activity in another type 3 copper protein, namely hemocyanin, which normally functions as an oxygen carrier. The nature of the conformational changes induced in type 3 copper proteins by the denaturant SDS is unknown. This comparative study demonstrates that arthropod hemocyanins can be converted from being an oxygen carrier to a form which exhibits phenoloxidase activity by incubation with SDS, with accompanying changes in secondary and tertiary structure. Structural characterisation, using various biophysical methods, suggests that the micellar form of SDS is required to induce optimal conformational transitions in the protein which may result in opening a channel to the di-copper centre allowing bulky phenolic substrates access to the catalytic site.
Keywords: Abbreviations; Hc; hemocyanin; PO; phenoloxidaseHemocyanin; Phenoloxidase; Spectroscopy; Enzyme activation; Isothermal titration calorimetry
N1, N12-Diacetylspermine oxidase from Debaryomyces hansenii T-42: Purification, characterization, molecular cloning and gene expression
by Mikio Bakke; Kazuhiko Shimoji; Naoki Kajiyama (pp. 1395-1401).
An FAD-dependent N1, N12-diacetylspermine oxidase (DASpmOX), which seems suitable for enzymatic determination of the tumor marker N1, N12-diacetylspermine (DASpm), was isolated from Debaryomyces hansenii T-42. DASpmOX exhibited the most excellent specificity toward DASpm among all polyamine oxidases found to date, and the specificity for DASpm could be raised by adjusting the pH of the buffer and adding TritonX-100. In potassium phosphate (pH 7.0) with 0.3% TritonX-100, this enzyme did not have any detectable activity toward free polyamines, and the reaction rate of N1, N8-diacetylspermidine, N1-acetylspermine, N1-acetylspermidine, and N8-acetylspermidine was only 19%, 7.8%, 7.8%, and 1.0% of that of DASpm, respectively. The gene encoding DASpmOX was cloned and expressed in Escherichia coli. The apparent kcat and Km values of recombinant enzyme for DASpm were found to be 158 s−1 and 3.1×10−4 M under the conditions described above, respectively.
Keywords: Abbreviations; DASpmOX; N; 1; ,; N; 12; -diacetylspermine oxidase; DASpm; N; 1; ,; N; 12; -diacetylspermine; DASpd; N; 1; ,; N; 8; -diacetylspermidine; N; 1; -ASpd; N; 1; -acetylspermidine; Spm; spermine; Spd; spermidine; SSAT; spermine/spermidine; N; 1; -acetyltransferase; PAO; polyamine oxidase; CAO; copper-containing amine oxidase; PADH; polyamine dehydrogenase; N; 8; -ASpd; N; 8; -acetylspermidine; N; 1; -ASpm; N; 1; -acetylspermine; Put; putrescine; ELISA; enzyme-linked immunosorbent assay; TOOS; N; -ethyl-; N; -(2-hydroxy-3-sulfopropyl)-; m; -toluidine; POD; peroxidase; BSA; bovine serum albumin; BPAO; bovine plasma amine oxidase; PMSF; phenymethylsulfonyl fluoride; TLC; thin-layer chromatography; daspmo; N; 1; ,; N; 12; -diacetylspermine oxidase gene; CyDTA; trans; -1,2-diaminocyclohexane-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid; EGTA; O; ,; O; ′-Bis(2-aminoethyl)ethyleneglycol-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid; Me-EDTA; 1,2-diaminopropane-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid; N; 1; -ASpdOX; N; 1; -acetylspermidine oxidaseDiacetylspermine oxidase; Debaryomyces hansenii; Substrate specificity; Cloning; Heterologous expression
Hypoxia influences the cellular cross-talk of human dermal fibroblasts. A proteomic approach
by Federica Boraldi; Giulia Annovi; Fabio Carraro; Antonella Naldini; Roberta Tiozzo; Pascal Sommer; Daniela Quaglino (pp. 1402-1413).
The ability of cells to respond to changes in oxygen availability is critical for many physiological and pathological processes (i.e. development, aging, wound healing, hypertension, cancer). Changes in the protein profile of normal human dermal fibroblasts were investigated in vitro after 96 h in 5% CO2 and 21% O2 (pO2=140 mm Hg) or 2% O2 (pO2=14 mm Hg), these parameters representing a mild chronic hypoxic exposure which fibroblasts may undergo in vivo. The proliferation rate and the protein content were not significantly modified by hypoxia, whereas proteome analysis demonstrated changes in the expression of 56 proteins. Protein identification was performed by mass spectrometry. Data demonstrate that human fibroblasts respond to mild hypoxia increasing the expression of hypoxia inducible factor (HIF1a) and of the 150-kDa oxygen-regulated protein. Other differentially expressed proteins appeared to be related to stress response, transcriptional control, metabolism, cytoskeleton, matrix remodelling and angiogenesis. Furthermore, some of them, like galectin 1, 40S ribosomal protein SA, N-myc-downstream regulated gene-1 protein, that have been described in the literature as possible cancer markers, significantly changed their expression also in normal hypoxic fibroblasts. Interestingly, a bovine fetuin was also identified that appeared significantly less internalised by hypoxic fibroblasts. In conclusion, results indicate that human dermal fibroblasts respond to an in vitro mild chronic hypoxic exposure by modifying a number of multifunctional proteins. Furthermore, data highlight the importance of stromal cells in modulating the intercellular cross-talk occurring in physiological and in pathologic conditions.
Keywords: Human fibroblast; Primary cell culture; Hypoxia; Connective tissue; Proteome; 2D gel electrophoresis; Mass-spectrometry
Impacts of Cd(II) on the conformation and self-aggregation of Alzheimer's tau fragment corresponding to the third repeat of microtubule-binding domain
by Ling-Feng Jiang; Tian-Ming Yao; Zhi-Liang Zhu; Chong Wang; Liang-Nian Ji (pp. 1414-1421).
Environmental exposure to some heavy metals such as cadmium appears to be a risk factor for Alzheimer's disease (AD), however, definite mechanism of their toxicity in AD remains to be elucidated. Previous studies largely focused on the metal ions binding to β-amyloid, however, very few papers concerned the interaction between tau and metal ions. For the first time, we investigated the impacts of Cd(II) on the conformation and self-aggregation of Alzheimer's tau peptide R3, corresponding to the third repeat of microtubule-binding domain. The initial state of R3 was proven to be dimeric linked by intermolecular disulfide bond, in the non-reducing buffer (Tris–HCl buffer pH7.5, containing no reducing reagent). In this paper, we show that Cd(II) can accelerate heparin-induced aggregation of R3 or independently induce the aggregation of R3, as monitored by ThS fluorescence. In the presence of Cd(II), the resulting R3 filaments became much smaller, as revealed by electron microscopy. Binding to the Cd(II) ion, the dimeric R3 partially lost its random coil, and converted to α-helix structure, as revealed by CD and Raman spectrum. Stoichiometric analysis of CD signal against the ratio of [Cd(II)]/[R3] suggested that the coordination intermediate consisted of two R3 dimers binding to a central cadmium ion. As the seed, the coordination intermediate could extensively accelerate the self-aggregation of R3 via promoting the nucleation step. On the other hand, gain in α-helix structure on the peptide chain, by coordinating with Cd(II), could be a critical role to promote self-aggregation, as revealed by Raman spectrum. These results provide a further insight into the mechanism of tau filament formation and emphasize the possible involvement of Cd(II) in the pathogenesis of AD.
Keywords: Abbreviations; AD; Alzheimer's disease; PHF; paired helical filament; MAP; microtubule-binding protein; 3RMBD; three-repeat microtubule-binding domain; 4RMBD; four-repeat microtubule-binding domain; MBD; microtubule-binding domain; R1; first repeat peptide of the four-repeat microtubule-binding domain; R2; second repeat peptide of the four-repeat microtubule-binding domain; R3; third repeat peptide of the four-repeat microtubule-binding domain; R4; fourth repeat peptide of the four-repeat microtubule-binding domain; ThS; thioflavin S; EM; electron microscopy; CD; circular dichroism; HPLC; high-pressure liquid chromatography; TFA; trifluoroacetic acidCadmium; Alzheimer's disease; tau Peptide; Aggregation
Purification, cloning and characterization of a novel peroxidase isozyme from sweetpotatoes ( Ipomoea batatas)
by Annette Rompel; Michael Albers; Joseph I. Naseri; Carsten Gerdemann; Klaudia Büldt-Karentzopoulos; Beate Jasper; Bernt Krebs (pp. 1422-1430).
An anionic peroxidase from sweetpotato tubers is purified and characterized. The isozyme ibPrx15 is purified to homogeneity by affinity chromatography using a concanavalin A column. The isoelectric point was determined to p I 4.9. MALDI-MS detected a singly charged molecule with a mass of 42029 Da. Absorption spectra of ibPrx15 compounds I, II and III were obtained after treatment with H2O2 at room temperature. Comparative data of ibPrx15 on substrate specificity to tobacco anionic peroxidase (TOP) and horseradish peroxidase (HRP) reveal similar specific activity towards a series of conventional substrates except for iodide, which is a two-electron donor interacting directly with the compound I derivative in the catalytic cycle. ibPrx15 exhibits a high specific activity towards iodide about 103-fold to that of tobacco peroxidase. The amino acid sequence of the main isozyme ibPrx15 was determined by Edman degradation and by sequencing the amplified cDNA fragments. ibPrx15 has 86% identity to another Ipomoea sequence ibPrx05 and 72% identity with a sequence from Populus trichocarpa (PtPrx72).
Keywords: Abbreviations; ibPOD; peroxidase isolated from sweetpotatoes (; Ipomoea batatas); TOP; tobacco peroxidase; HRP; horseradish peroxidase; rpm; rotations per minute; HPLC; high-pressure liquid chromatography; MALDI-MS; matrix-assisted laser desorption/ionization mass spectrometry; Prx; peroxidase; PCR; polymerase chain reaction; RACE; rapid amplification of cDNA ends; fw; forward; bw; backward; TFA; trifluoroacetic acid; RZ; Reinheitszahl; ABTS; 2,2′-azino-; bis; -(3-ethyl-6-benzothiazolinsulphonate)Peroxidase; Sweetpotato; Ipomoea batatas; cDNA; Sequence
Mutation of the H-helix in antithrombin decreases heparin stimulation of protease inhibition
by Patrick R. Gonzales; Timothy D. Walston; Laureano O. Camacho; Dana M. Kielar; Frank C. Church; Alireza R. Rezaie; Scott T. Cooper (pp. 1431-1437).
Blood clotting proceeds through the sequential proteolytic activation of a series of serine proteases, culminating in thrombin cleaving fibrinogen into fibrin. The serine protease inhibitors (serpins) antithrombin (AT) and protein C inhibitor (PCI) both inhibit thrombin in a heparin-accelerated reaction. Heparin binds to the positively charged D-helix of AT and H-helix of PCI. The H-helix of AT is negatively charged, and it was mutated to contain neutral or positively charged residues to see if they contributed to heparin stimulation or protease specificity in AT. To assess the impact of the H-helix mutations on heparin stimulation in the absence of the known heparin-binding site, negative charges were also introduced in the D-helix of AT. AT with both positively charged H- and D-helices showed decreases in heparin stimulation of thrombin and factor Xa inhibition by 10- and 5-fold respectively, a decrease in affinity for heparin sepharose, and a shift in the heparin template curve. In the absence of a positively charged D-helix, changing the H-helix from neutral to positively charged increased heparin stimulation of thrombin inhibition 21-fold, increased heparin affinity and restored a normal maximal heparin concentration for inhibition.
Keywords: Abbreviations; AT; antithrombin; PCI; protein C inhibitor; TM; thrombomodulinThrombin; Factor Xa; Thrombomodulin; Coagulation; Serpin
β-PrP form of human prion protein stimulates production of monoclonal antibodies to epitope 91–110 that recognise native PrPSc
by Azadeh Khalili-Shirazi; Maria Kaisar; Gary Mallinson; Samantha Jones; Daljit Bhelt; Carol Fraser; Anthony R. Clarke; Simon H. Hawke; Graham S. Jackson; John Collinge (pp. 1438-1450).
Prion diseases are associated with accumulation of strain-dependent biochemically distinct, disease-related isoforms (PrPSc) of host-encoded prion protein (PrPC). PrPSc is characterised by increased β-sheet content, detergent insolubility and protease resistance. Recombinant α-PrP adopts a PrPC-like conformation, while β-PrP conformationally resembles PrPSc, to these we raised 81 monoclonal antibodies in Prnp 0/0 mice. The N-terminal residues 91–110 are highly immunogenic in β-PrP-immunised mice and of (17/41) anti-β-PrP antibodies that could be epitope-mapped, ∼70%, recognised this segment. In contrast, only 3/40 anti-α-PrP antibodies could be mapped and none interacted with this region, instead recognising residues 131–150, 141–160 and 171–190. Native PrPC was recognised by both antibody groups, but only anti-β-PrP antibodies directed to 91–110 residues recognised native PrPSc with high affinity, where in addition, species heterogeneity was also evident. Within the six anti-β-PrP antibodies studied, they all recognised PK-treated native human and mouse PrPSc, four failed to recognise PK-treated native bovine PrPSc, one of which also did not recognise native PK-treated ovine PrPSc, showing the epitope becomes exposed on unfolding and disaggregation. These results demonstrate strain-dependent variations in chain conformation and packing within the 91–110 region of PrPSc.
Keywords: Antibody; BSE; CJD; Prion; PrP; Scrapie
Examination of the mechanism and energetic contribution of leaving group activation in the purine-specific nucleoside hydrolase from Trypanosoma vivax
by John N. Barlow; Jan Steyaert (pp. 1451-1461).
The mechanism and energetics of the purine-specific nucleoside hydrolase from Trypanosoma vivax ( TvNH) are examined by stopped-flow at low temperatures. TvNH is shown to follow an ordered uni–bi kinetic mechanism and high forward commitment with inosine as substrate ( Cf=1.9±0.6). Measurement of partitioning of the Michaelis complex, which exists at negligible concentrations in the steady state, is achieved using a novel sequential-mixing stopped-flow method. A product burst is observed with p-nitrophenyl riboside (pNPR) in the pre-steady state, indicating that a step after chemistry rate determines kcat. Comparison of the kinetics of inosine and pNPR turnover shows that the dominant energetic contribution towards catalysis in TvNH comes from ribosyl and water activation (11 kcal/mol); however, leaving group activation still makes a considerable (8 kcal/mol) contribution. A solvent isotope effect (D2O k=1.7) on the chemistry transient τ1 with guanosine as substrate was observed. Therefore, the leaving group is unlikely to be protonated prior to N-glycosidic bond cleavage. We propose that leaving group protonation is, by itself, unlikely to account for the large energetic contribution of leaving group activation. Instead, we postulate that active site binding interactions to the purine leaving group are required for efficient ribosyl and/or water activation.
Keywords: Abbreviations; NH; nucleoside hydrolase; Tv; NH; nucleoside hydrolase from; Trypanosoma vivax; Cf; NH; nucleoside hydrolase from; Crithidia fasciculata; TbbNH; nucleoside hydrolase from; Trypanosoma brucei brucei; pNPR; p; -nitrophenylriboside; Guo; guanosine; Ino; inosine; NP; nucleoside phosphorylase; XO; xanthine oxidase; 7MG; 7-methylguanosine; τ; 1; transientTransient kinetic; Nucleoside hydrolase; Solvent isotope effect; Forward commitment
Effect of salt on the activity of Streptomyces prolyl aminopeptidase
by Misugi Uraji; Jiro Arima; Yoshiko Uesugi; Masaki Iwabuchi; Tadashi Hatanaka (pp. 1462-1469).
A salt-tolerant prolyl aminopeptidase from Streptomyces aureofaciens TH-3 (TH-3PAP) was purified from a culture supernatant. The gene encoding TH-3PAP was cloned and sequenced. The primary structure of TH-3PAP showed 65% identity with that of PAP from Streptomyces lividans (SLPAP) and possessed a conserved catalytic motif, GxSxGG, which is conserved in the α/β hydrolase fold family. The characterization of the recombinants TH-3PAP and SLPAP indicated a difference: in 4.0 M NaCl, TH-3PAP showed enzyme activity, whereas SLPAP was inactive. Next, we constructed chimeras between TH-3PAP and SLPAP using an in vivo DNA shuffling system and a sandwich chimera (sc-PAP), whose region from 63 to 78 amino acids of TH-3PAP was substituted with that of SLPAP. Comparison of the biochemical properties between TH-3PAP and the salt-sensitive sc-PAP suggested that the fine tuning of the N-terminal conformation of TH-3PAP by hydrophobic interaction is important for the salt tolerance mechanism of the enzyme.
Keywords: Abbreviations; PAP; prolyl aminopeptidase; TH-3PAP; PAP from; Streptomyces aureofaciens; TH-3; SLPAP; PAP from; Streptomyces lividans; E; .; Escherichia; RIBS; repeat-length-independent and broad-spectrum; pNA; p; -nitroanilide; r-; recombinant; c-PAP; chimeric PAP; sc-PAP; sandwich chimera; ANS; 8-anilino-1-naphthalenesulfonate; CD; circular dichroism; XPAP; PAP from; Xanthomonas campestris; PCR; polymerase chain reaction; DIG; digoxigenin; Gm; gentamycin; Sm; streptomycin; Cm; chloramphenicolProlyl aminopeptidase; Streptomyces; Salt tolerance
Biophysical investigation of human heparan sulfated-glucosaminyl 3-O-sulfotransferase-3A: A mutual effect of enzyme oligomerisation and glycosaminoglycan ligand binding
by Iris Wille; Angelika Rek; Evelyn Krenn; Andreas J. Kungl (pp. 1470-1476).
3-O-sulfation of heparan sulfate (HS) is the rarest modification within heparan sulfate biosynthesis resulting in unique biological activities. Heparan sulfated-glucosaminyl 3-O-sulfotransferase-3A (3-OST-3A) (EC 2.8.2.23) generates a binding site for the envelope glycoprotein D (gD) of herpes simplex virus 1. We have expressed the sulfotransferase domain of the human heparan sulfate 3-OST-3A isoform in Escherichia coli and subsequently purified the active enzyme which was found to be present as an oligomer under nonreducing conditions. The activity of the enzyme was tested by a novel gD-dependent gel mobility assay. A biophysical characterisation of 3-OST-3A was performed to study ligand binding and ligand-induced structural changes. Interestingly, the natural substrate HS did not cause a secondary structural change in the enzyme, whereas heparin and chondroitin sulfate did, both of which also exhibited similar high affinity binding to 3-OST-3A compared to HS as detected by isothermal fluorescence titrations. In cross-link assays, only HS was found to induce high molecular aggregates of 3-OST-3A whereas other GAG ligands did not or even inhibited enzyme oligomerisation like the K5 polysaccharide, which was nevertheless found to bind to the enzyme. We therefore conclude that since 3-OST-3A is able to bind also non-substrate GAG ligands with high affinity, discrimination among ligands is triggered by protein oligomerisation.
Keywords: Abbreviations; HS; heparan sulfate; GAG; glycosaminoglycan; HSPG; heparan sulfate proteoglycan; GlcNAc; N; -acetylglucosamine; GlcNS; N; -sulfated glucosamine; HSV-1 gD; herpes simplex virus type 1 glycoprotein D; r-3-OST-3A; recombinant 3-O-sulfotransferase (G148-G406); CD; circular dichroism; PAPS; adenosin 3′-phosphate 5′-phosphosulfate; CS; chondroitin sulfateFluorescence; Circular dichroism; Folding; Enzyme; Biosynthesis
The protein profile of mouse mature cumulus–oocyte complex
by Yan Meng; Xiao-hui Liu; Xiang Ma; Ya Shen; Lu Fan; Jing Leng; Jia-Yin Liu; Jia-Hao Sha (pp. 1477-1490).
In mammals, the cumulus–oocyte complex (COC) is the main component of ovarian follicles. Bi-directional communication between oocytes and surrounding cumulus granulosa cells is essential for the development of oocytes and ovarian follicles. In this study, we performed proteomic profiling of mouse mature COC, using two-dimensional gel electrophoresis and mass spectrometry. A total of 259 protein spots were identified, which correspond to 156 individual proteins. We also discovered some protein families, which may play important roles in ovarian follicular development. Immunostaining was conducted to determine the subcellular localization of specific proteins from selected protein families of interest, and to examine their expression patterns during follicle development. These data provide valuable information for future studies to identify proteins involved in ovarian follicular development and related reproductive abnormalities.
Keywords: Mouse cumulus–oocyte complex; Two-dimensional electrophoresis; Mass spectrometry; Protein profile
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