Alzheimer disease is the most common causeof dementia, causing by founded a high densityof amyloid plaque in the brain tissue of victims and Amyloid beta-protein is 40to42 residuepolypeptide that forms the plaque core in Alzheimer disease. when I comparisonbetween 4 techniques ;high performance chromatography, immunoprecipitation ,size exclusion chromatography, ELISA each one can detect amyloid beta proteinput in spesefic HPLCand immunoprecipitation show best result and more accurateand more sensitive . Keywords: Alzheimer’sdisease, amyloid beta protein, immunoprecipitation, size exclusionchromatography, ELISA, HPLC Coupled with Tandem Mass Spectrometry 1. Introduction:Alzheimer disease is the most common causeof dementia, causing by founded a high densityof amyloid plaque in the brain tissue of victims. (Hely, Reid, Adena, Halliday, & Morris, 2008) .
The mean of dementia is related to lossof intellectual and social skills. The patient with this disease forgetsimportant people in their live(Alexander & Larson, 2007). The main factors that cause Alzheimer disease are genetic, lifestyleand environmental factors; these factors affect the brain overtime by damagethe brain cell (neurons).(Lin, Epel, & Blackburn, 2012)Mechanism of Alzheimer disease: The brainconsists of tens of billions of neurons; these neurons are responsible forprocess and transmit information via electron and chemical signals as specificAlzheimer disease play role in damage these neurons so the process of send messagesbetween different part of the brain and from the brain to the muscle and organsof the body are disrupt .(D’Amelio & Rossini, 2012) .
Mitochondria that provided the energydemand of the cell; damage the cell by producing reactiveoxygen species (ROS) in the some case, thus leads to cellular oxidative stress.The risk factors of (AD): diabetes, hypertension, obesity, smoking,depression, low educational attainment and physical inactivity. (Barnes & Yaffe, 2011) Symptoms of (AD) initially begin withforget the names of places and objects and often change in mood .in the middlestage symptoms of these disease is difficult to remember the names of peoplethey know and may struggle to recognize their family and friends. in theadvanced stage of these disease, the symptoms become more progressive includeHallucinations and delusions may come and go over the course of the illness,but can get worse as the condition progresses .In addition to difficulty eatingand swallowing (dysphasia) (Association, 2013).
Amyloid beta-protein is 40to42 residuepolypeptide that forms the plaque core in Alzheimer disease (Arispe, Rojas, & Pollard, 1993) ,(Zheng & Koo, 2006),. The two best known forms of the A? peptideare known as A?1-40 and A?1-42 depending on the number of amino acids in the peptide. The common cause of cognitive failure in older humans remainscontroversial; because of the difficulty in providing direct mechanisticevidence that an amyloid-? species impairs cognitive function. Therefore Theaim of this study is to detect amyloid proteins in Alzheimer disease by severaltechniques: size exclusion chromatography, high performance liquidchromatography (HPLC), ELISA and immuneprecipitation; because The rate ofprogression varies greatly; People with Alzheimer’s live an average of8-20years so the course of the disease depends in part on age at diagnosis andwhether a person has other health conditions. Alzheimer’s disease is stillincurable and the mortality rate is increasing rapidly and Reasons for there increased are not established (Todd, Barr, Roberts,& Passmore, 2013). It is very important to learn more about the more efficienttechniques to detect amyloid beta proteins also A? monomers, oligomers oramyloid plaque is the primary toxic species is not known.
On the other hand sometherapeutic strategies are aimed to inhibition of ?- and ?-secretases toprevent formation of A? while others are depend on reducing A? peptides throughimmunological. Hardy, John(Hardy & Selkoe, 2002), (Das, 2012), (Gelfanova et al., 2007)1.Immunoprecipitation (IP)Immunoprecipitation (IP) is a populartechnique that uses the high specificity of antibodies that is immobilized to asolid support such as magnetic particles or agarose resin (Nilsson, Halim, Grahn, & Larson, 2013); theseantigens binding to that particular protein and these method used in manyscientific fields. The aim of these techniques is used to isolate andconcentrate a particular protein from a sample containing wide range differentproteins. In fact the antibody: antigen complex is removed with the aidof an antibody be coupled to a solid substrate at some point in the procedure..(McManus, Lamoliatte, & Thibault, 2017) Materials and methods thatwere used in immunoprecipitation techniques: were develop protocol based on thecharacteristics of the amyloid fold, such as its resistance to proteolysis andits capacity to be recognized by specific conformational antibodies in order topurify amyloid from organisms,.
We were used a two-step strategy withproteolytic digestion as the first step followed by immunoprecipitation usingthe amyloid conformational antibody LOC which is useful for isolating A?1–40amyloid fibrils therefore, the results they reached were by produced amyloidfibrils by used A?1–40 ;which is the peptide associated with Alzheimer’s disease.So that IP approach was efficient at pulling down exogenously added A?1–40fibrils from wild type worm PDS, we investigated a more physiological conditionusing tissue homogenates from a C. elegans AD model and our strategy that were useswas useful for immunoprecipitating A? amyloid fibrils produced in vitro and invivo and that by the use of this approach we detected more aggregates in olderworms when compared to young worms. The strategies they used were effective indeveloping a sensitive assay for the purification and detection of amyloidfibrils and the LOC antibody that were used was efficient in imunoprecipitating A? fibrils produced in vivo and themethod that used was useful for the purification of amyloid beta protein frombiological samples.
The results you came up that the immunoprecipitation usingthe LOC antibody is useful for isolating A?1–40 amyloid fibrils, on the otherhand it fails to capture fibrils of other amyloidogenic proteins, such as?-synuclein and gelsolin. (Greiner, Kelly, & Palhano, 2014) 2. Highperformance liquid chromatography (HPLC) Coupled with Tandem Mass SpectrometryHighPerformance Liquid Chromatography (HPLC) is a form of column chromatographythat pumps analyte in the mobile phase at high pressure through a column withstationary phase. The sample is carried by a moving gas stream of helium ornitrogen. HPLC has the ability to separate, and identify compounds that arepresent in any sample that can be dissolved in a liquid in trace concentration. (Mitulovi? & Mechtler, 2006)To determinequantitation of A? peptides using high-performance liquid chromatographycoupled with tandem mass spectrometry in a linear ion trap mode.
Materials thatwere used in HPLC system consist of RP-HPLC was performed using C8 column (3.0mm × 150 mm) with a particle size of 3.5?m. A 10-?L aliquot of synthetic A?1–40was injected onto the HPLC column. The mobile phase consisted of solvent A (5%acetonitrile, 5% isopropanol, 10 mM ammonium acetate, and 90% water, pH = 7)and solvent B (45% acetonitrile, 45% isopropanol, 10 mM ammonium acetate, and10% water, pH = 7). A?1–40 peptide was eluted using a gradient elution programof 20% to 85% B in 15 min, a 5-min hold at 85% B, followed by a return to 20% Bfor a 10-min equilibration. The flow rate was 0.4 mL/min.
Eluate from theRP-HPLC column was split to both the UV detector and electrospray ionization MSsource. (ChenganDu, Corinne Ramaley, Hugh McLean, Susan C Leonard, & Jeff Miller, 2005) While in themass spectrometry experiments were performed on either a hybridquadrupole/linear ion trap 4000 Q TRAP MS/MS system or Q TRAP MS/MS system. Allanalyses were performed in the highly sensitive linear ion trap (LIT) mode. Thetransition from a multiply charged molecular ion was used to detect andquantitate A?1–40 peptide using the Q TRAP system in the positive electrosprayionization mode.
Reversed-phase HPLC is a well-establishedtechnique for the analysis of peptides and proteins in various biologicalmatrices.(Gygi et al., 1999) . However,A?1–40 is a very nonpolar peptide with poor recovery in conventionalreversed-phase HPLC with an acidic mobile phase.
The result were that HPLCelution provided excellent separation of A?1–40 peptide with a mixture ofisopropanol/acetonitrile/water/10 mM ammonium acetate (pH = 7).The A?1–40standard calibration curves show excellent linearity from 34 ng to 2500 ngA?1–40 of column sample load. The LC/MS/MS method using a conventional RP-HPLCcolumn coupled with the Q TRAP system in the Enhanced Product Ion scanning modewas capable of detecting A?1–40 at low nanogram levels with good specificityand linearity of response. The transition of multiply charged molecular ions ofproteins or peptides to a singly charged product ion provided a useful tool toqualitatively and quantitatively analyze peptides.
Multiple reactionsmonitoring in the triple quadrupole mode is commonly used in quantitation usingLC-MS/MS analysis these is a useful by using a single transition and severalsummed transitions is that it allows detection and quantitation of severaltarget compounds simultaneously. The combination of reversed-phased HPLC withmass spectrometry had been previously reported using a 75-nm-diameter nano-HPLCcolumn coupled with a single-quadrupole mass spectrometer in the selective ionmonitoring mode using multiply charged ions of A?1–40.The detectionlimit in the present communication is 31.25 ng in column load using a3.0-mm-diameter conventional C8 column. This level of sensitivity is far fromsufficient for the direct analysis of A? peptide in biological fluids (0.25ng). but the detection limit could be potentially enhanced up to 1600-fold toreach 0.
02-ng levels, by adapted current method to a 75-nm-diameter nanoflowHPLC column.The product ion scan mode was chosen in order to achieve superiorspecificity in A?1–40 quantitation instead of the selective ion monitoring modebecause may not achieve the specificity required for quantitation of A?1–40 inpatient serum. Also the product ion scan mode provides superior specificity forA?1–40 identification and quantification compared with selective ion monitoringin the selective ion scan mode. So that these methods were establishing anaccurate and reliable analytical method to qualitatively and quantitativelyanalyze A? peptides.(C.Du, C. Ramaley, H.
McLean, S. C. Leonard, & J. Miller, 2005)3. Sizeexclusion chromatography These techniqueswere used A? species secreted by cultured cells, or by complex mixtures of A?assembly forms in the brains of APP transgenic mice. Used Aqueously soluble(Tris-buffered saline (TBS)) extracts underwent non-denaturing size exclusionchromatography (SEC). extracted soluble amyloid ?–protein (A?)oligomers directly from the cerebral cortex of typical AD subjects .First, massspectrometry of the 4 and 8 kDa bands IP’d from the GuHCl extract of AD cortexconfirmed that each contained tryptic peptides of human A? .
Second, IP of thisextract with an A?40-specific antibody (2G3) and WB with an A?42-specificantibody (21F12) revealed an A?40/42 heterodimer migrating at 8 kDa. Performingthis co-IP using 21F12 for both IP and WB yielded a much stronger dimer signal,indicating that most of the 8 kDa species are A?42/42 homodimers.(Shankar et al., 2008).soluble A?exists in various assemblies, with the smallest native oligomer being a dimerthese result indicate that Soluble dimers are the smallest A? assembly form inhuman brain to acutely perturb synapse physiolo. SEC experiments show that soluble A? dimersinhibit LTP, it remained possible that a small molecule from human brain wasbound to the A? dimers and was required to impair long term potentiation LTP. Toaddress this possibility, we were generated a synthetic A?40 peptide in whichserine 26 was mutated to cysteine (A?40-S26C). An A? dimer was observed uponoxidation, and this inhibited LTP nearly 20-fold more potently than didwild-type synthetic A?40.
This pure, synthetic dimer cannot contain any otherfactors present in AD TBS, establishing that A? dimers alone are sufficient toperturb synapse physiology.wehomogenized TBS-insoluble pellets of plaque-rich AD cortex in 2% SDS in orderto insoluble amyloid cores isolated from AD cortex without inhibit hippocampalLTP.addition ofintact cores to the ACSF perfusate did not affect LTP (139.4 ± 7.6%).Therefore, in physiologic buffer (ACSF), amyloid cores do not acutely releasesoluble A? dimers to alter synaptic plasticity.
IP/WB analyses revealed that A?dimers also were not released from amyloid cores incubated in physiologicalbuffers at 37°C for 24 hr, suggesting that highly insoluble A? aggregates suchas amyloid plaque cores represent dimer-rich structures that do not readilydissociate. We used non-denaturing gel filtration coupled withIP/WB and subsequent immunodepletion or neutralization with epitope-specific A?antibodies to ascribe the pathogenic effects to soluble A? oligomers,principally dimers. Our findings fulfill an essential requirement forestablishing disease causation in AD so that the soluble A? oligomers extractedfrom AD brains potently impair synapse structure and function and that dimersare the smallest synaptotoxic species.4.
ELISAenzyme-linked immunosorbent assayTest usescomponents of the immune system (antibodies involves at least one antibody withspecificity for a particular antigen) and color chemicals to the identify asubstance. Examples of the uses of an ELISA test include diagnosing infectionssuch as HIV and some allergic diseases like food allergies.(Xia et al., 2009)Primarilyused for the detection of proteins (small molecules and ions such as glucoseand potassium). The substances detected by ELISA tests can include hormones,viral antigens (dengue fever, for example), bacterial antigens (TB, forexample), and antibodies that the body has made in response to infection. we wereestablished a sensitive and specific oA? ELISA to analyze oA? species in humansamples.
we utilized a synthetic A? peptide, A?1–40 Ser26Cys Toconfirm the specificity of the ELISA. new ELISA accurately measuredthe levels of dA? in a linear fashion and is highly sensitive to the dissociationof dA? to monomers because ?ME, dissociation of the disulfide bond markedlyreduced the amount of dA? signal, and the levels of remaining dA? were lessthan 10% of the same fraction without ?ME. Detection of the synthetic dimersonly in the absence of ?-mercaptoethanol clearly demonstrated the specificityof our oA? ELISA, with far less ability to detect monomers. ouroA?-specific ELISA allowed us to establish a close quantitative relationshipbetween the levels of A?1–42 and oA? in human plasma and brain tissues.oA? ELISAprovides an accurate method to measure such species in human blood, our resultsdo not yet validate any one A? species as a biomarker for AD. difficult toobtain a clear separation of AD from control subjects simply by measuring thelevels of plasma A? species . However, our findings suggest that measuringplasma A? and oA? over 1–2 years or more can reveal a significant reduction inplasma A?, especially A?42, and this finding raises the possibility of a directrelationship of plasma A? to brain amyloid formation. So that Thespecificity of oA? ELISA was validated with a disulfide crossed-linked,synthetic A?1–40Ser26Cys dimer that was specifically detected before but notafter the dissociation of the dimers in ?-mercaptoethanol.
Plasma assays showedthat relative oA? levels were closely associated with relative A?42 monomerlevels across all subjects. Analysis of sequential plasma samples from a subsetof the AD patients, including a patient with AD caused by a presenilinmutation, revealed decreases in both oA? and A?42 monomer levels over a 1–2year period. In brain tissue from 9 AD and 7 control subjects, both oA? andmonomeric A?42 were consistently higher in the AD case.AnoA?-specific ELISA reveals a tight link between oA? and A?42 monomer levels inplasma and brain, and both forms can decline over time in plasma, presumablyreflecting their increasing insolubility in the brain. Conclusion: immunoprecipitation : using the LOC antibody isuseful for isolating A?1–40 amyloid fibrils, on the other hand it fails tocapture fibrils of other amyloidogenic proteins, such as ?-synuclein andgelsolin;sothat IP approach was efficient at pulling down exogenously addedA?1–40 fibrils from wild type worm PDS and also by using LOC antibody.on theother hand High performance liquid chromatography (HPLC) Coupled with TandemMass Spectrometry techniques included the HPLC elution providedexcellent separation of A?1–40 peptide and excellent linearity from 34 ng to2500 ng A?1–40 of column sample load and by chosen product ion scan mode we were achieve superior specificity in A?1–40quantitation So that these methods were establishing an accurate and reliableanalytical method to qualitatively and quantitatively analyze A? peptides. in SEC experiments show that soluble A? dimersinhibit LTP, SEC fractions of AD TBS containing either higher MW complexes (ADSEC 4), native A? dimers (AD SEC 8) or monomers (AD SEC 10) .
and in the Elisa the oA?-specific ELISA reveals a tight link between oA?and A?42 monomer levels in plasma and brain. studies about HPLC have found that theretention behavior and recovery of A? peptide can be improved when the columnis eluted under basic conditions However, in the present studies, enhancedproduct ion scans using a linear ion trap mode was chosen as the quantitationtool due to its 5- to 10-fold higher sensitivity than multiple reactionmonitoring.RP-HPLC coupled with MS/MS demonstrated a significant sensitivityenhancement over conventional RP-HPLC.C8 capillary RP-HPLC column (0.
3 mm × 150mm), a 50-fold sensitivity enhancement was achieved for A?1–40 peptidequantitation, which is slightly less than the theoretical 100-fold improvementpredicted in going from a 3.0-mm-diameter conventional column to a 0.3-mmcapillary column.
The less than ideal improvement in sensitivity reflects thosefactors that will compromise the performance of capillary and nanoflow HPLC,such as dead volumes, gradient delay, etc.Work is inprogress to transfer the established HPLC-MS/MS method to a nanoflow HPLC-MS/MSsystem, to directly determine the A?1–40 concentrations in blood serum samplesfrom both patients with Alzheimer’s disease and normal controls. The analyticalconditions presented here will also be adapted to analyze A?1–42, a morehydrophobic and, consequently, a more challenging amyloid peptide forquantitation in biological fluids.The directquantitation of A?1–40 in biological fluids using HPLC-MS/MS may provide a newavenue to identify possible biomarkers for the .The method presented in this communicationcan be adapted to nanoflow HPLC-MS/MS to further enhance the sensitivity up to1000-fold for direct quantitation of A?1–40 in blood samples development ofAlzheimer’s disease.