Protective Effect of Mangosteen Extract against β-Amyloid-Induced Cytotoxicity, Oxidative Stress and Altered Proteome in SK-N-SH Cells Primchanien Moongkarndi,† Chatchawan Srisawat,‡ Putita Saetun,§ Jiraporn Jantaravinid,‡ Chayanon Peerapittayamongkol,‡ Rungtip Soi-ampornkul,‡ Sarawut Junnu,‡ Supachok Sinchaikul,| Shui-Tein Chen,|,⊥ Patcharakajee Charoensilp,‡ Visith Thongboonkerd,*,§,# and Neelobol Neungton*,‡ Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand, Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, Institute of Biological Chemistry and Genomic Research Center, Academia Sinica, Taipei, Taiwan, Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan, and Center for Research in Complex Systems Sciences, Mahidol University, Bangkok, Thailand Received July 15, 2009
β-amyloid (Aβ) plays a key role in the pathogenesis of Alzheimer’s disease (AD) by inducing neurotoxicity and cell death mainly through production of reactive oxygen species (ROS). Garcinia mangostana L. (mangosteen) has been recognized as a major source of natural antioxidants that could decrease ROS. However, its role in protection of Aβ-induced cytotoxicity and apoptosis in neuronal cells remains unclear. We therefore examined such a protective effect of mangosteen extract (ME) by evaluating cell viability using MTT test, ROS level, caspase-3 activity, and cellular proteome. Treating SK-N-SH cells with 5-20 µM Aβ(1-42) for 24 h caused morphologically cytotoxic changes, decreased cell viability and increased ROS level, whereas preincubation with 50-400 µg/mL ME 30 min before the induction by Aβ(1-42) successfully prevented such cytotoxic effects in a dose-dependent manner (completely at 400 µg/mL). The Aβ-induced increase in caspase-3 activity was also preventable by 400 µg/mL ME. Proteomic analysis using 2-D gel electrophoresis (n ) 5 gels/group) followed by mass spectrometry revealed 63 proteins whose levels were significantly altered by Aβ(1-42) induction. Interestingly, changes in 10 proteins were successfully prevented by the ME pretreatment. In summary, we report herein the significant protective effects of ME against Aβ-induced cytotoxicity, increased ROS, and increased caspase activity in SK-N-SH cells. Moreover, proteomic analysis revealed some proteins that might be responsible for these protective effects by ME. Further characterizations of these proteins may lead to identification of novel therapeutic targets for successful prevention and/or decreasing the severity of AD. Keywords: Alzheimer’s disease • amyloid • cytotoxicity • mangosteen extract • proteome • proteomics
Introduction Alzheimer’s disease (AD) is the most common cause of dementia affecting approximately 10-15% of elderly at the age * To whom correspondence should be addressed. Neelobol Neungton, Department of Biochemistry, 13th Fl. - Chudhadhuj Bldg., Siriraj Hospital, 2 Prannok Rd., Bangkoknoi, Bangkok 10700, Thailand. Tel: +66-2- 4198583. Fax: +66-2-4111428. E-mail:
[email protected]. Visith Thongboonkerd, Medical Proteomics Unit, Office for Research and Development, 12th Fl. Adulyadejvikrom Bldg., Siriraj Hospital, 2 Prannok Rd., Bangkoknoi, Bangkok 10700, Thailand. Tel/Fax: +66-2-4184793. E-mail:
[email protected] or
[email protected]. † Department of Microbiology, Faculty of Pharmacy, Mahidol University. ‡ Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University. § Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University. | Institute of Biological Chemistry and Genomic Research Center, Academia Sinica. ⊥ Institute of Biochemical Sciences, College of Life Science, National Taiwan University. # Center for Research in Complex Systems Sciences, Mahidol University.
2076 Journal of Proteome Research 2010, 9, 2076–2086 Published on Web 03/17/2010
over 65 years.1 The incidence of dementia is doubling every 5 years afterward and reaches >50% by the age over 85 years.1,2 AD is more serious at present because people, all over the world, can now live longer. Generally, the disease starts with an insidious onset of neuronal loss, followed by gradually progressive deterioration of brain function and finally death within approximately 10 years after the diagnosis. Currently, the definite cause remains unknown and there is no successful treatment or prevention of AD. The hallmark found in AD patients is accumulation of neurofibrillary tangles and senile plaques in the brain. The main component of senile plaques is β-amyloid peptide (Aβ), which plays a major role in the development of AD.3,4 This polypeptide, consisting of 39-43 amino acid residues, is proteolytically cleaved from a transmembrane amyloid precursor protein (APP) by β- and γ-secretases.5 Many in vitro and in vivo studies have demonstrated Aβ-induced toxicity as an early and responsible event in the pathogenesis of AD.6-8 In cell culture, Aβ can directly induce cell death and cause neurons vulnerable to excitotoxicity and 10.1021/pr100049v
2010 American Chemical Society
Protective Effect of Mangosteen Extract against β-Amyloid oxidative insults. Aβ facilitates generation of free radicals, which cause peroxidation of membrane lipids, and increases the production of reactive oxygen species (ROS), resulting in cell damage and apoptosis.8 Recently, many efforts have been made on searching for the antioxidants that could reduce Aβ toxicity in AD. Among these, polyphenols that are natural substances obtained from plants, fruits and vegetables have drawn most of attention from the investigators.9 Garcinia mangostana L. (Mangosteen) is a tropical evergreen tree that grows well in Southeast Asia, particularly in Myanmar, Sri Lanka, and Thailand. In Asia, mangosteen is named as the “Queen of Fruits” due to its pleasant taste. The fruit hull of mangosteen has been used as a traditional medicine for many years for treatment of skin infection, wound, dysentery, and diarrhea. Many studies have reported that the pericarp of mangosteen contains xanthone, mangostin, tannin, chrysanthemin, garcinone, gartanin, vitamin B1, B2, C and other bioactive substances. Xanthone and its derivatives are classified as polyphenolic compounds, which are present with high amounts in the pericarp of mangosteen. They are reported as potent chemicals for anti-inflammatory, antitumor, antioxidant, and antibacterial activities.10,11 Additionally, their potent scavenging activities on nitric oxide, hydroxyl and superoxide radicals have been reported. Moreover, the water-soluble partition of methanol-extract of mangosteen pericarp has a neuroprotective effect against oxidative stress in various neuronal cells in vitro and in vivo.12,13 However, there is no previous report on the inhibitory or protective effect of mangosteen extract (ME) on Aβ-induced neurotoxicity. Thus, the aim of the present study was to investigate the protective effect of ME against Aβ-induced toxicity in neuronal cells by evaluating cell viability using MTT test, ROS level, caspase-3 activity, and cellular proteome.
Materials and Methods Cell Culture and Induction of Cytotoxicity by Aβ. SK-NSH human neuroblastoma cells (HTB-11) (ATCC; Manassas, VA) were cultured in minimum essential medium (MEM) (GIBCO, Invitrogen Corporation; Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum, 15% (w/v) sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 100 µg/mL streptomycin and 100 units/ml penicillin G, and were maintained in a humidified incubator at 37 °C with 5% CO2. Aβ(1-42) (American Peptide Company; Sunnyvale, CA) was dissolved in 5% NH4OH to make 1 mM stock solution and stored at -20 °C until use. Various doses (5-20 µM) of Aβ1-42 were added to the cells and further incubated for 24-48 h. Preparation and Administration of Mangosteen Extract (ME). Mangosteens were collected from a local farm in Chantaburi province, Thailand. After careful washing, the pericarp was removed, air-dried for 5 days, and then crushed into powder using a regular plant grinding machine. Thereafter, the pericarp powders (1 kg) were macerated with methanol (5 L) at 60 °C. The crude extract was then filtered through Whatman No.1 filter paper under vacuum and stored at 4 °C for 2 days (to allow the settle down of the unwanted precipitate; but should not keep longer, which could introduce bacterial contamination). Thereafter, the solvent was concentrated by vacuum centrifugation on a vacuum rotary evaporator. The concentrated crude extract was then partitioned with ethylacetate (EtOAc) and water to separate the active constituents based on their polarity. Both partitions were preliminarily analyzed for certain chemicals (i.e., xanthone derivatives,
research articles garcinone D, alpha mangostin, and gamma mangostin) by densitometric thin layer chromatography (TLC) and highperformance liquid chromatography (HPLC). The water partition used in the present study should contain total phenolic compounds ranging from 150-200 mg/g (of gallic acid equivalent as determined by Folin-Ciocalteau method). The amount of R-mangostin in the extract, when determined by TLC, must be 60 for peptide mass fingerprinting “or” peptide ions scores >40 for MS/MS analyses. Western Blot Analysis. Western blot analysis was performed to confirm the proteomic data. Equally 20 µg total protein extracted from each sample was resolved in each lane of SDSPAGE at 150 V for approximately 2 h using SE260 mini-Vertical Electrophoresis Unit (GE Healthcare). After the completion of SDS-PAGE, proteins were transferred onto a nitrocellulose membrane and nonspecific bindings were blocked with 5% milk in PBS for 1 h. The membrane was then incubated with mouse monoclonal antikaryopherin β1 (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA) or mouse monoclonal antiGAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibody (Santa Cruz Biotechnology, Inc.) (both at a dilution of 1:1,000 in 5% milk/PBS) at 4 °C overnight. After washing, the membrane was further incubated with rabbit antimouse IgG conjugated with horseradish peroxidase (1:2000 in 5% milk/PBS) (Dako; Glostrup, Denmark) at room temperature for 1 h. Reactive protein bands were then visualized with SuperSignal West Pico chemiluminescence substrate (Pierce Biotechnology, Inc.; Rockford, IL). Statistical Analysis. All the quantitative data are reported as Mean ( SEM. Statistical analyses were carried out using SPSS version 16.0 (SPSS Inc.; Chicago, IL). Multiple comparisons of more than two groups of variables were performed by oneway analysis of variance (ANOVA) with Dunnett’s posthoc test, whereas comparisons between two groups of variables were performed by unpaired Student’s t test (when the data distributed normally) or nonparametric Mann-Whitney U test (when the data did not distribute normally). P values less than 0.05 were considered as statistical significant.
Results Cell Viability and Cytotoxicity. The cytotoxic effect of Aβ(1-42) was determined by MTT reduction. MTT is a tetrazolium salt that can be reduced to formazan by mitochondrial dehydrogenase, which is active only in living cells. Aβ(1-42) at any doses significantly reduced the viability of SK-N-SH cells (Figure 1A). At 24 h of incubation, the cytotoxic effect of Aβ(1-42) was dosedependent. Overall, 5-20 µM Aβ(1-42) significantly reduced the cell viability to approximately 54.0-68.5% as compared to the untreated control cells. Morphological study showed cell shrinkage and membrane blebbing, a typical feature of apoptotic cell death, under inverted microscope (data not shown).
Protective Effect of Mangosteen Extract against β-Amyloid
Figure 1. Cytotoxicity of Aβ(1-42) (A) and protective effect of ME (B) in SK-N-SH cells examined by MTT test. In (B), various doses of ME were preincubated with the cells 30 min prior to induction with 10 µM Aβ(1-42) for 24 h. The data are reported as Mean ( SEM (n ) 12 independent experiments for each bar). * ) p < 0.05 compared to the untreated control; # ) p < 0.05 compared to the induction with 10 µM Aβ(1-42) without ME preincubation.
Induction of cytotoxicity by Aβ(1-42) at 10 µM for 24 h was then used for all subsequent experiments to evaluate the protective effect of ME. Preincubation of SK-N-SH cells with 50-400 µg/ mL ME for 30 min successfully prevented the cytotoxic effect of Aβ(1-42) (the data reached significant levels at g100 µg/mL, and the Aβ-induced cytotoxicity was completely prevented at 400 µg/mL) (Figure 1B). ME at the dosage of 400 µg/mL was then used for all subsequent experiments to examine the protective effect of ME against unpleasant events induced by Aβ. Intracellular ROS Level. The ROS production was evaluated by measuring intracellular ROS level, which reflects the oxidative stress in SK-N-SH cells upon exposure to Aβ. Aβ(1-42) at 5-20 µM caused significant increase of intracellular ROS level in a dose-dependent manner (approximately 1.7, 4.8 and 11.5fold increase at the dosages of 5, 10, and 20 µM, respectively) (Figure 2A). Preincubation of SK-N-SH cells with 400 µg/mL ME significantly prevented the increase of intracellular ROS induced by 10-20 µM Aβ(1-42) (Figure 2B). The data also demonstrate that ME alone had no effect on intracellular ROS level (comparable to the untreated control cells). Caspase-3 Activity. The caspase-3 activity was normalized and expressed as a fold-change as compared to the untreated control cells. Induction with 10 µM Aβ(1-42) caused approximately 2.3-fold increase in caspase-3 activity, whereas a preincubation of SK-N-SH cells with 400 µg/mL ME successfully and completely prevented the increase of caspase-3 activity (remained at its basal level) (Figure 3). The data also demon-
research articles
Figure 2. (A) Intracellular ROS level using the DCF-DA assay to measure DCF, which reflects ROS level. ROS level was increased by induction with Aβ(1-42) in a dose-dependent manner. (B) Aβ(1-42)-induced increase of ROS level was successfully prevented by preincubation of the cells with 400 µg/mL ME. The data are reported as Mean ( SEM (n ) 4 independent experiments for each bar). * ) p < 0.05 compared to the untreated control; # ) p < 0.05 compared to the induction with Aβ(1-42) without ME preincubation.
Figure 3. Effects Aβ(1-42) and ME on caspase-3 activation. The data are report as Mean ( SEM of ratios of caspase-3 activities in experimental groups as compared to the untreated control (n ) 5 independent experiments for each bar). * ) p < 0.05 compared to the untreated control; # ) p < 0.05 compared to the induction with 10 µM Aβ(1-42) without ME preincubation.
strate that ME alone had no effect on caspase-3 activity (comparable to the untreated control cells). Proteome Profiling. Effect of Aβ(1-42) on SK-N-SH neuronal cells was also evaluated by proteome profiling. The 2-D proteome profiles of untreated control and Aβ-treated cells are illustrated in Figure 4A and B, respectively. Exposure to 10 µM Aβ(1-42) caused marked alterations in the proteome profile of SK-N-SH cells with 63 protein spots whose abundance levels Journal of Proteome Research • Vol. 9, No. 5, 2010 2079
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Figure 4. Altered cellular proteome profile by induction with 10 µM Aβ(1-42). Totally 100 µg total proteins derived from untreated control cells (A) or those exposed to 10 µM Aβ(1-42) for 24 h (B) were resolved with 2-DE and visualized by Deep Purple fluorescence dye (n ) 5 gels/group derived from individual cultured flasks). Spot matching and quantitative intensity analysis revealed significant changes in abundance levels of 63 protein spots (labeled with numbers, which correspond to those reported in Table 1). These altered proteins were then identified by Q-TOF MS and/or MS/MS analyses (see Table 1).
Figure 5. Protective effect of 400 µg/mL ME against Aβ(1-42)-induced changes in cellular proteome. Totally 100 µg total proteins derived from (A) untreated control cells, (B) those exposed to 10 µM Aβ(1-42) for 24 h, and (C) those preincubated with 400 µg/mL ME 30 min prior to induction with Aβ(1-42) were resolved with 2-DE and visualized by Deep Purple fluorescence dye (n ) 5 gels/group derived from individual cultured flasks). Only Aβ(1-42)-induced changes that were successfully prevented by preincubation with 400 µg/mL ME are labeled. Details of such preventable changes (ID, quantitative data, and statistics) are summarized in Table 1.
were significantly altered by Aβ(1-42). Among these, 21 protein spots had increased levels (1.29-16.95 folds), 39 protein spots had decreased levels (0.11-0.77 folds), 2 spots (#412 and #485) were absent in the Aβ-treated cells, and 1 spot (#1244) was present only in the Aβ-treated cells (Table 1). These altered proteins were then identified by Q-TOF MS and/or MS/MS analyses. While most of the altered proteins (33 proteins) were identified only by peptide mass fingerprinting, 3 other proteins were identified only by MS/MS and 20 proteins were identified by both peptide mass fingerprinting and MS/MS analyses. Note that there were still other 7 altered proteins, which might be low abundant component, remained unidentified. This might be simply due to their amounts, which were below the threshold for identification by MS and MS/MS analyses. We also evaluated the protective effect of ME on Aβ-induced alterations in cellular proteome of SK-N-SH cells. The data demonstrate that preincubation with 400 µg/mL ME prior to induction by 10 µM Aβ(1-42) successfully prevented some 2080
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changes in the cellular proteome (Figure 5). The magnified images of these altered proteins, which were preventable by ME pretreatment, are illustrated in Figure 6A. Among these, changes in 7 proteins were completely preventable, whereas alterations in 3 proteins were partially prevented (Table 1 and Figure 6B). Western Blot Analysis. To confirm the proteomic data, we performed Western blot analysis of karyopherin β1. The proteomic data showed a decrease in level of this protein after an exposure to 10 µM Aβ(1-42) that was preventable by pretreatment with 400 µg/mL ME (Table 1 and Figure 6). Western blot analysis also revealed the consistent results and confirmed the proteomic data (Figure 7).
Discussion Aβ has been reported to be toxic to neurons through various mechanisms including ROS generation, mitochondrial dysfunc-
Protective Effect of Mangosteen Extract against β-Amyloid
research articles
Figure 6. (A) Zoom-in images and (B) quantitative intensity data of protein spots whose changes induced by 10 µM Aβ(1-42) were successfully prevented by 400 µg/mL ME. The data were obtained from 5 individual gels (derived from 5 independent cultured flasks) per group. C ) complete protection; P ) partial protection.
tion and apoptosis.14,15 Aβ(1-42), antiparallel β-pleated sheet peptide, is a major constituent of diffused plaques in AD brain. Many reports have documented that Aβ(1-42), after cleavage from amyloid precursor protein (APP), is an important factor in the pathogenesis of AD.3,4 In addition, the increased production of Aβ(1-42) in familial AD patient accelerates neurodegeneration, probably because of the higher propensity of Aβ(1-42) for amyloid fibrillogenesis, resulting in formation of bioactive amyloid plaque species.6,7,16 In vitro and transgenic mice studies have shown that Aβ(1-42) is much more amyloidogenic than Aβ(1-40).17 We therefore employed Aβ(1-42) to examine the Aβ-induced cytotoxicity in SK-N-SH neuronal cells throughout our present study. SK-N-SH is a human neuroblastoma cell line developed by Biedler et al. in 197318 and has been extensively
employed in many of cell-mediated cytotoxicity assays. Unlike a primary culture, which is usually a mixture of different cells (e.g., microglial cells, fibroblasts); SK-N-SH cells allow direct investigation of cytotoxicity in a single cell type. We have clearly demonstrated the cytotoxic effect of Aβ(1-42) on SK-N-SH cells using MTT assay, a widely used test for cell viability. The yellow tetrazolium salt was reduced into purple formazan by an enzyme dehydrogenase localized in the mitochondria of viable cells. Our data indicated that Aβ(1-42) induced cytotoxicity in a dose-dependent manner (Figure 1A). These results were consistent with those reported in previous studies, demonstrating that Aβ(1-42) at µM concentration levels caused apoptosis in culture cells.19 Journal of Proteome Research • Vol. 9, No. 5, 2010 2081
2082
Journal of Proteome Research • Vol. 9, No. 5, 2010
485
473 478 482
421 431 463 472
408 412
405
393
391
371
311 323 324 332 340 348
109 137 154 173 199 215 290 294
108
spot no.
protein name
Dynein heavy chain 5, axonemal (Ciliary dynein heavy chain 5) (Axonemal beta dynein heavy chain 5) Unidentified Unidentified Unidentified Ubiquitin-activating enzyme E1 Actinin, alpha 4, isoform CRA_a Karyopherin beta 1 Adenylosuccinate lyase 1 78 kDa glucose-regulated protein precursor (GRP 78) (Immunoglobulin heavy chain binding protein) (BiP) Keratin 1 Serum albumin Serum albumin Hypothetical protein isoform 3 Aminopeptidase B Transforming, acidic coiled-coil containing protein 3 isoform 1 TERF1 (TRF1)-interacting nuclear factor 2 Heterogeneous nuclear ribonucleoprotein K Chaperonin containing TCP1, subunit 6A isoform a Tubulin, alpha, ubiquitous isoform 19 Thioredoxin reductase GRIM-12 Protein phosphatase 5, catalytic subunit isoform 2 Tubulin, beta 2C, isoform CRA_b FK506-binding protein 4 Septin 6 Heterogeneous nuclear ribonucleoprotein H (hnRNP H) isoform 3 Hypothetical protein FLJ25778 HLA-B associated transcript 1 Protein disulfide isomerase-related protein 5 RuvB-like 2 isoform 5
NCBI ID
identified by
MS
MS
MS
MS, MS/MS MS MS MS MS MS
NA NA NA MS MS MS, MS/MS MS MS, MS/MS
MS
gi|114678321
gi|51094792 gi|4758112 gi|1710248
gi|119608775 gi|114642856 gi|78369494 gi|73970377
gi|3820535 gi|114677997
MS, MS/MS
MS MS, MS/MS MS, MS/MS
MS, MS/MS MS, MS/MS MS MS, MS/MS
MS MS
gi|109096498 MS, MS/MS
gi|4502643
gi|55958547
gi|152941126
gi|11935049 gi|28592 gi|28592 gi|109019206 gi|10933784 gi|108994967
NA NA NA gi|23510338 gi|119577213 gi|19923142 gi|126338737 gi|73968070
gi|149732989
149, 57
82, NA 104, 29 115, 138
239, 220 171, 145 68, NA 103, 191
88, NA 62, NA
185, 199
133, NA
76, NA
96, NA
86, 211 145, NA 75, NA 74, NA 102, NA 74, NA
NA, NA NA, NA NA, NA 92, NA 178, NA 124, 12 74, NA 237, 170
75, NA
identification scores (MS, MS/MS)
Table 1. Summary of Altered Proteome in SK-N-SH Cells Induced by 10 µM Aβ(1-42)a
43, 3
14, NA 32, 2 40, 14
55, 13 46, 12 29, NA 39, 9
29, NA 30, NA
58, 17
45, NA
24, NA
29, NA
25, 8 33, NA 22, NA 20, NA 24, NA 14, NA
NA, NA NA, NA NA, NA 22, NA 31, NA 30, 1 31, NA 46, 7
8, NA
%cov (MS, MS/MS)
18, 1
16, NA 16, 1 13, 4
26, 5 19, 3 9, NA 12, 3
13, NA 9, NA
20, 5
18, NA
7, NA
11, NA
13, 4 18, NA 12, NA 15, NA 13, NA 11, NA
NA, NA NA, NA NA, NA 15, NA 23, NA 21, 1 11, NA 26, 3
30, NA
no. of matched peptides (MS, MS/MS)
0.72 0.38 0.45 0.54 0.50 0.52 0.62 0.67
NA NA 0.1443 ( 0.0114 0.1044 ( 0.0074 NA NA 0.0823 ( 0.0169 0.0312 ( 0.0035 NA NA 0.1172 ( 0.0077 0.0526 ( 0.0084 5.49 118.86 0.0750 ( 0.0027 0.0406 ( 0.0022 5.27 105.30 0.0616 ( 0.0045 0.0311 ( 0.0038 4.68 98.42 0.1673 ( 0.0205 0.0876 ( 0.0057 8.49 56.82 0.0444 ( 0.0023 0.0273 ( 0.0056 5.09 72.25 0.4748 ( 0.0354 0.3195 ( 0.0255
0.41
0.0852 ( 0.0082 0.2618 ( 0.0191
0.0440 ( 0.0054 0.3837 ( 0.0359
45.32 2.5484 ( 0.1742 1.0495 ( 0.1354
5.33
47.19
0.1040 ( 0.0070
0.0000 ( 0.0000
0.00
0.19 1.49 0.76
9.25 140.12 0.0360 ( 0.0021 0.0070 ( 0.0070 5.44 49.42 0.1720 ( 0.0156 0.2560 ( 0.0132 4.95 46.51 0.8002 ( 0.0283 0.6093 ( 0.0441
55.32 51.00
0.40 1.38 0.45 1.55
4.88 5.49 6.35 5.89
6.36 5.57
49.25 2.2091 ( 0.1299 0.8729 ( 0.0477 47.33 0.1461 ( 0.0173 0.2018 ( 0.0159 49.09 0.1030 ( 0.0185 0.0462 ( 0.0074 48.52 0.0608 ( 0.0065 0.0942 ( 0.0125
1.94
0.68 0.00
58.44
42.01
0.0332 ( 0.0028 0.0386 ( 0.0043
0.68
0.0307 ( 0.0028
0.0550 ( 0.0053 39.18
0.56
0.0568 ( 0.0046 0.2486 ( 0.0556 0.3919 ( 0.1188 0.0400 ( 0.0048 0.1283 ( 0.0161 0.0033 ( 0.0033
0.0752 ( 0.0053 0.0314 ( 0.0027 0.0231 ( 0.0026 0.0618 ( 0.0079 0.0549 ( 0.0022 0.0307 ( 0.0035 66.20 71.32 71.32 118.66 73.26 90.94
0.0226 ( 0.0033 0.0000 ( 0.0000
4.94
6.23
5.43
9.10
8.16 6.05 6.05 5.29 5.51 5.33
0.75 7.92 16.95 0.65 2.34 0.11
0.45
Aβ
533.21
0.0387 ( 0.0015
5.82
control
ratio (Aβ/ Control)
0.0867 ( 0.0082
pI
MW (kDa)
intensity (mean ( SEM)
P value
No No No No No No
No No No No No Complete No No
Partial Complete No No
