Both Targeted Mass Spectrometry and Flow Sorting Analysis Methods Detected the Decreased Serum Apolipoprotein E Level in Alzheimer's Disease Patients
2013; Elsevier BV; Volume: 13; Issue: 2 Linguagem: Inglês
10.1074/mcp.m113.028639
ISSN1535-9484
AutoresSun-Ho Han, Jun Seok Kim, Young-Ju Lee, Hee-Sun Choi, Jong‐Won Kim, Duk L. Na, Eun Gyeong Yang, Myeong‐Hee Yu, Daehee Hwang, Cheolju Lee, Inhee Mook‐Jung,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoApolipoprotein E (ApoE) polymorphism has been appreciated as a valuable predictor of Alzheimer disease (AD), and the associated ε4 allele has been recognized as an indicator of susceptibility to this disease. However, serum ApoE levels have been a controversial issue in AD, due to the great variability regarding the different target detection methods, ethnicity, and the geographic variations of cohorts. The aim of this study was to validate serum ApoE levels in relation to AD, particularly using two distinct detection methods, liquid chromatography–selected reaction monitoring (SRM) mass spectrometry and microsphere-based fluorescence-activated cell sorting (FACS) analysis, to overcome experimental variations. Also, comparison of serum ApoE levels was performed between the level of protein detection by FACS and peptide level by SRM in both control and AD patients. Results from the two detection methods were cross-confirmed and validated. Both methods produced fairly consistent results, showing a significant decrease of serum ApoE levels in AD patients relative to those of a control cohort (43 control versus 45 AD, p < 0.0001). Significant correlation has been revealed between results from FACS and SRM (p < 0.0001) even though lower serum ApoE concentration values were measured in protein by FACS analysis than in peptide-level detections by SRM. Correlation study suggested that a decrease of the serum ApoE level in AD is related to the mini-mental state exam score in both results from different experimental methods, but it failed to show consistent correlation with age, gender, or clinical dementia rating. Apolipoprotein E (ApoE) polymorphism has been appreciated as a valuable predictor of Alzheimer disease (AD), and the associated ε4 allele has been recognized as an indicator of susceptibility to this disease. However, serum ApoE levels have been a controversial issue in AD, due to the great variability regarding the different target detection methods, ethnicity, and the geographic variations of cohorts. The aim of this study was to validate serum ApoE levels in relation to AD, particularly using two distinct detection methods, liquid chromatography–selected reaction monitoring (SRM) mass spectrometry and microsphere-based fluorescence-activated cell sorting (FACS) analysis, to overcome experimental variations. Also, comparison of serum ApoE levels was performed between the level of protein detection by FACS and peptide level by SRM in both control and AD patients. Results from the two detection methods were cross-confirmed and validated. Both methods produced fairly consistent results, showing a significant decrease of serum ApoE levels in AD patients relative to those of a control cohort (43 control versus 45 AD, p < 0.0001). Significant correlation has been revealed between results from FACS and SRM (p < 0.0001) even though lower serum ApoE concentration values were measured in protein by FACS analysis than in peptide-level detections by SRM. Correlation study suggested that a decrease of the serum ApoE level in AD is related to the mini-mental state exam score in both results from different experimental methods, but it failed to show consistent correlation with age, gender, or clinical dementia rating. Alzheimer disease (AD) 1The abbreviations used are: ADAlzheimer diseaseAATAATVGSLAGQPLQERApoEapolipoprotein EAUCarea under the curveCDRclinical dementia ratingCEcollision energyDPdeclustering potentialFACSfluorescence-activated cell sortingSRMselected reaction monitoringLGPLGPLVEQGRMFImean fluorescent intensityMMSEmini-mental state examPEphycoerythrinROCreceiver operating characteristic. 1The abbreviations used are: ADAlzheimer diseaseAATAATVGSLAGQPLQERApoEapolipoprotein EAUCarea under the curveCDRclinical dementia ratingCEcollision energyDPdeclustering potentialFACSfluorescence-activated cell sortingSRMselected reaction monitoringLGPLGPLVEQGRMFImean fluorescent intensityMMSEmini-mental state examPEphycoerythrinROCreceiver operating characteristic. is the most common neurodegenerative disorder among the elderly population and causes fatal cognitive dysfunction, including learning and memory impairment. Two distinctive features of AD, senile plaques of amyloid-β and neurofibrillary tangles in the brain, have been considered as hallmarks of the main pathological phenomena in AD (1Hardy J.A. Higgins G.A. Alzheimer's disease: the amyloid cascade hypothesis.Science. 1992; 256: 184-185Crossref PubMed Scopus (5063) Google Scholar, 2Hardy J. Allsop D. Amyloid deposition as the central event in the aetiology of Alzheimer's disease.Trends Pharmacol. Sci. 1991; 12: 383-388Abstract Full Text PDF PubMed Scopus (1837) Google Scholar, 3Hardy J. Selkoe D.J. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.Science. 2002; 297: 353-356Crossref PubMed Scopus (10962) Google Scholar). An increased number of AD patients in modern society leads to multiple problems—not only physical and emotional problems, but also societal problems regarding employment status and financial crises in the public health system (4Alloul K. Sauriol L. Kennedy W. Laurier C. 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Alzheimer disease AATVGSLAGQPLQER apolipoprotein E area under the curve clinical dementia rating collision energy declustering potential fluorescence-activated cell sorting selected reaction monitoring LGPLVEQGR mean fluorescent intensity mini-mental state exam phycoerythrin receiver operating characteristic. Potential risk factors for AD discovered thus far include older age, female gender, low level of education, and smoking (8Launer L.J. Andersen K. Dewey M.E. Letenneur L. Ott A. Amaducci L.A. Brayne C. Copeland J.R. Dartigues J.F. Kragh-Sorensen P. Lobo A. Martinez-Lage J.M. Stijnen T. Hofman A. Rates and risk factors for dementia and Alzheimer's disease: results from EURODEM pooled analyses. EURODEM Incidence Research Group and Work Groups. European Studies of Dementia.Neurology. 1999; 52: 78-84Crossref PubMed Google Scholar). Apolipoprotein E (ApoE) polymorphism has been recognized as the most probable indicator to date (9Corder E.H. Saunders A.M. Strittmatter W.J. 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Increased plasma apolipoprotein E (apoE) levels in Alzheimer's disease.Neurosci. Lett. 1997; 223: 29-32Crossref PubMed Scopus (96) Google Scholar). In contrast, a Rotterdam study by Slooter et al. reported no increase in the serum ApoE level in AD as determined via a colorimetric method (22Slooter A.J. de Knijff P. Hofman A. Cruts M. Breteler M.M. Van Broeckhoven C. Havekes L.M. van Duijn C.M. Serum apolipoprotein E level is not increased in Alzheimer's disease: the Rotterdam study.Neurosci. Lett. 1998; 248: 21-24Crossref PubMed Scopus (56) Google Scholar). The ApoEurope project collected patient and control samples from nine centers in eight European countries and demonstrated both increased occurrence of the ApoE ε4 allele and decreased serum ApoE levels in AD via ApoE quantification using an immunoturbidimetric method (23Siest G. Bertrand P. Qin B. Herbeth B. Serot J.M. Masana L. Ribalta J. Passmore A.P. Evans A. Ferrari M. Franceschi M. Shepherd J. Cuchel M. Beisiegel U. 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Multiplexed analysis of human cytokines by use of the FlowMetrix system.Clin. Chem. 1998; 44: 2057-2060Crossref PubMed Scopus (138) Google Scholar). Each functional bead has variable intensities of two fluorophores and is identified by a certain combination of fluorescence intensities. The beads can be conjugated with capture antibody against different protein targets for multiplex detection. A target protein in a sample can be captured on the surface of a functional bead by binding to the capture antibody. Biotinylated detection antibody for ApoE can be applied to measure the target protein concentration via phycoerythrin (PE)-streptavidin treatment. Compared with the conventional ELISA method, microsphere-based fluorescence detection technology has advantages of high reproducibility, small sample amount, high sample capacity, and multiplex application of various target proteins (37Gordon R.F. McDade R.L. Multiplexed quantification of human IgG, IgA, and IgM with the FlowMetrix system.Clin. Chem. 1997; 43: 1799-1801Crossref PubMed Scopus (61) Google Scholar, 38Oliver K.G. Kettman J.R. Fulton R.J. Multiplexed analysis of human cytokines by use of the FlowMetrix system.Clin. Chem. 1998; 44: 2057-2060Crossref PubMed Scopus (138) Google Scholar, 39Prabhakar U. Eirikis E. Davis H.M. Simultaneous quantification of proinflammatory cytokines in human plasma using the LabMAP assay.J. Immunol. Methods. 2002; 260: 207-218Crossref PubMed Scopus (168) Google Scholar). In this study, we attempted to characterize the AD-related changes in serum ApoE levels using two distinct modes of detection: protein detection via microsphere-based FACS analysis and peptide detection via LC-SRM mass spectrometry. The results from the two methods were cross-validated, and experimental variations for serum ApoE quantification were minimized. Both results presented fairly consistent results, showing a significant decrease of serum ApoE levels in AD patients relative to those of a control cohort (43 control versus 45 AD, p < 0.0001). Blood samples were collected from patients with AD (n = 45) and age-matched nondemented elderly control subjects (n = 43) at Samsung Medical Center (Seoul, Korea) with approval from the Institutional Ethical Review Board. All subjects consented to the study, and informed consent was obtained from each entrant. AD patients were diagnosed using criteria for probable AD established by the National Institute of Neurological and Communicative Disorders and the Stroke-Alzheimer's Disease and Related Disorders Association (40McKhann G. Drachman D. Folstein M. Katzman R. Price D. Stadlan E.M. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease.Neurology. 1984; 34: 939-944Crossref PubMed Google Scholar). Clot-activator-treated polypropylene vacuum capillary tubes (Vacuplus®, Medigene, Seoul, Korea) were used for the collection of blood samples. The tubes were incubated at room temperature for 30 min, allowing fibrinogen in the blood to aggregate, and then centrifuged at 1,100g for 15 min to separate fibrinogen aggregates and other cellular components. The supernatant was collected and frozen at −80 °C until use, and no freeze-and-thaw cycle was applied before use. Measurement of serum ApoE level was performed using a BDTM Human Soluble Protein Master Buffer Kit (Catalog No. 558265, BD Biosciences-PharMingen, San Diego, CA), and a BDTM Functional Bead Conjugation Buffer Set (Catalog No. 558556, BD Biosciences, San Diego, CA). Anti-human apolipoprotein E mAb (Catalog No. 3712-3-1000, Mabtech AB, Nacka Strand, Sweden) was used as a capture antibody, and biotinylated anti-human apolipoprotein E mAb (Catalog No. 3712-6-1000, Mabtech AB, Nacka Strand, Sweden) was used as a detection antibody. First, beads were conjugated with capture antibody for ApoE. For covalent conjugation to functional beads, antibodies were modified by sulfosuccinimidyl 4-N-maleimidomethyl cyclohexane 1-carboxylate (Catalog No. 22322, Thermo Scientific, Rockford, IL) and filtered using a Bio-Rad Spin Column (Catalog No. 732-6231, Bio-Rad, Hercules, CA). Beads were incubated with dithiothreitol (Catalog No. 15397, USB, Cleveland, OH), and conjugation was performed with modified antibodies ended by N-ethylmaleimide (Catalog No. 23030, Thermo Scientific, Rockford, IL). Proper conjugation of the capture antibody to functional beads was confirmed using PE-coupled goat anti-mouse IgG detector treatment (Catalog No. 558550, BD Biosciences, San Diego, CA), followed by the detection of PE fluorescence at 575 nm by flow cytometry. Beads were validated for proper increase of the PE fluorescence intensity according to ApoE concentration. Calibrator serum was used for validation and as an internal reference. The calibrator serum was a random mixture of multiple control serum samples, and an aliquot of calibrator was kept in a freezer. Human serum samples were diluted to 1:80 in assay buffer, and 6 × 103 beads/reaction were suspended in the capture bead diluent. Beads and serum samples were mixed together and incubated in the dark for 1 h at room temperature. Subsequently, biotinylated detection antibodies were added (200 ng/50 μl), and the samples were incubated in the dark for 2 h at room temperature. Then, 20 μl of streptavidin-PE (Catalog No. 349023, BD Biosciences, San Jose, CA) was added and incubated in the dark for 30 min at room temperature. The samples were washed and centrifuged, and bead pellets were suspended in 300 μl of wash buffer. A BD FACS Calibur flow cytometer (BD Biosciences, San Jose, CA) with CellQuest software was used for the detection of fluorescence after the calibration step with setup beads. The mean fluorescent intensity (MFI) of 300 events was used for the quantification of ApoE protein. The MFI of a negative control was subtracted from the MFI of the target protein in a sample. Two negative controls containing only diluents and fetal bovine serum (Catalog No. SH30406.02, Thermo Scientific, Tauranga, New Zealand) were used. Then, this background-corrected MFI was normalized by that of the calibrator serum as follows: (MFI of test samples − MFI of FBS)/MFI of calibrator serum. Chemically synthesized crude peptides, LGPLVEQGR and AATVGSLAGQPLQER, were purchased from JPT Peptide Technologies Berlin, Germany. These were used to optimize the instrumental parameters and to identify targets in clinical sera in SRM. The peptides contained ∼50 nmol crude peptides with ∼70% purity. Stock solutions (∼50 nmol/200 μl) were prepared in 20% acetonitrile (≥98%, J.T. Baker, Phillipsburg, NJ) with 1% formic acid (≥98%, Merck, Darmstadt, Germany). 5 pmol/ml of each peptide was used for optimization. Stable isotope standards, also known as heavy peptide labeled C-terminal [13C6,15N4] arginine (>98%, 21st Century Biochemicals, Marlboro, MA), were purchased for absolute quantification. For each sequence, amino acid analysis was performed, and the absolute peptide amount was given by the vendor. All stable isotope standard peptides were spiked into sera prior to tryptic digestion. The concentrations of total protein in sera were measured via Bradford assay (Bio-Rad, Richmond, CA). Serial dilutions of 0, 0.2, 0.4, 0.6, 0.8, and 1 μg/μl of Bradford reagent (1 μg/μl) were prepared for the measurement, and 50- or 100-fold diluted serum was used for the quantification. 200 μg of total protein was diluted 10-fold with 25 mm ammonium bicarbonate (>99%, Sigma) prior to denaturation with a 1:1 ratio of 10% (w/v) sodium deoxycholate (>98%, Sigma). Heavy peptides (50 fmol) were spiked into the solution. The disulfide bonds of intact proteins were reduced by tris(2-carboxyethyl)phosphine (Thermo Fisher Scientific) and incubated in a Thermomixer (Eppendorf, Hamburg, Germany) for 40 min at 37 °C. To alkylate cysteins, the reactants were cooled to room temperature (25 °C), and iodoacetamide (>98%, Sigma) was added with a final concentration of 14 mm and mixed for 40 min at room temperature in dark conditions. The highly concentrated sodium deoxycholate was diluted 10-fold ( 99%, USB, Cleveland, OH). Trypsin digestion (sequencing-grade modified trypsin, Promega, Madison, WI) was performed at a concentration of 1/40 (enzyme/substrate) and followed by incubation at 37 °C for 16 h. The reaction was stopped by 0.1% TFA acidification reagent at pH < 3.0 (TFA, >99%, MercK, Darmstadt, Germany). An MCX cartridge (Waters, Milford, MA) was used to clean up the samples, and the elutions were frozen and dried via vacuum centrifugation. All the analytes were stored at 80 °C when not in use. We empirically optimized SRM parameters by directly infusing the crude peptides into a QTrap5500 hybrid line
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