Osman Adil Jamshed Chem 4101 9th December, 2011 PrPC PrPSc Reference: Prion structure, http://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%2010/Prion%20Structure.html (accessed Oct 25 2011)Prions Prions are misfolded form of proteins – beta sheets preferred over alpha helix - classified as infectious pathogens1. Responsible for fatal neurodegenerative diseases in mammals by modifying the secondary structure of cellular prion protein (PrPc) to infected scarpie prion protein (PrPSc) 1. Transmissible spongiform encephalopathies – the transmissible form of prion diseases – spongiform as it causes sponge like lesions in the brain2. Creutzfeldt – Jakob disease (CJD) in humans, Bovine spongiform encephalopathy (BSE) in cattle, Scarpie of sheep2. Infected brain tissue with lesion giving the tissue the appearance of a sponge. Reference: Richt. J.; Hall, S., PLoS Pathogens, 2008, 4, 9.Prions sequence (more than 20 different variants are possible). Reference: TSE in Humans, http://labs.ansci.illinois.edu/novakofski/BSE/Human_TSEs_Mutations.htm (accessed Dec 7, 2011)Analytical Problem Prions are extremely stable and resistant to chemical and physical agents like heat, acids, alkalis, detergents and enzymatic proteolysis1&2. It is a “slow virus” that is fatal and virtually undetectable until its too late2. Simple cooking of foodborne variant Creutzfeldt-Jakob disease infected meat products especially brain, does not stop the transmission1. CJD is a variant of BSE. Animals products used in making feed in some countries, like UK, can facilitate the spread of BSE, if infected. The low quantities of BSE prions relative to normal proteins in most samples and similarity between the two isoforms makes detection difficult. Possible mechanism for the conversion. Reference: Prusiner, S.; Proc. Natl. Acad. Sci.,1998, 95, pp. 13363–13383.Hypothesis The studies would include analyzing animal by-products used to make cattle feed, in regions (factories nearby) with reported prion outbreaks and places with no prion outbreak. The data will be compared to the safety precautions and regulations enforced by factories in the region. Analyte = PrPSc; Matrices = biological, brain and CNS tissue. • Detecting the spread of the BSE among cattle would prevent sudden outbreak of BSE epidemic in the future and prevent humans from contracting the foodborne JCD – a variant of BSE. StudiesSeparation techniques3,4&5 Technique Pros Cons Nanospray Liquid chromatography High sensitivity and separation selectivity, lower solvent consumption, works well with MS Slow - nanoliters/min flow rate, small loading capacity Capillary Isoelectric Focusing Good separation, high resolution, based on isoelectric points that are quite different for normal prions and infected prions Different pIs for different species’ prions, needs LIF detector with a fluorescent label, does not work well with MS Reverse phase liquid chromatography Readily available, relatively cheap, works well with MS. Requires high sample preparation, low sensitivity References : 3 Shkundina, I. S.; Ter-Avanesyan, M. D.; Biochemistry (Moscow), 2007, 72, 13, pp. 1519-1536 4Onisko, B.; Dynin, I.; Requena, J.; Silva, C.; Erickson, M.; Carter, J.; J. Am. Soc. Mass Spectrom., 2007, 18, 1070-1079. 5Ramsay, L.; Dickerson, J.; Dovichi, N.; Electrophoresis 2009, 30, 297–302Detection Methods4&5 Detectors Advantages Disadvantages MS/MS (Q-TOF) Detection up to attomole level with PITC in sample, highly selective, library available Not quantitative, Expensive, destructive, takes relatively longer time than triple quadrupole. LIF Detection possible up to yoctomole level, high sensitivity, Requires fluorescence or a fluorescent reagent, expensive, moderate selectivity, relatively lower signal to noise ratio. Diagram of Q-TOF mass spectrometer operating in MS (upper) and MS/MS mode (lower) modes. Reference: Dunn, M.; Industry assignment; http://www.colorado.edu/chemistry/chem5181/QSTAR%20Pulsar.pdf, (accessed Dec 6, 2011)Homogenates will be centrifuged for 10 min at 5000 x g at 4 oC. Brain or nervous tissue will be homogenized (HT1000 Potter homogenizer) in ice-cold PBS containing 0.5% Nonidet p-40 and 0.5% deoxycholate to give a 10% (w/v) suspension. Sample preparation4 Supernatants of brain or nervous tissue homogenates were aliquoted and stored at -80 oC. Before analysis, bovine trysin (3.3μg in 33 μL water) digestion will be carried out at 37 oC overnight. The digestion will be stopped by adding formic acid (2μL) to give a pH of 2.3. 4Onisko, B.; Dynin, I.; Requena, J.; Silva, C.; Erickson, M.; Carter, J.; J. Am. Soc. Mass Spectrom., 2007, 18, 1070-1079.NanoLC/ESI/Q-TOF MS/MS Applied Biosystems MDS SCIEX QStar Pulsar equipped with Proxeon Biosystems nanoelectrospray source was used for detection and general ESI process (image on the far right). ESI process Figure Q-TOF MS/MS Figure Reference: Hybrid quadrupole-TOF (Q-TOF) for MS/MS, http://www.spectroscopynow.com/coi/cda/detail.cda?id=11316&type=EducationFeature&chId=10&page=1 (accessed Dec 6, 2011) Reference: Prions formation http://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%2010/Prion%20Structure.html (accessed Oct 25 2011)Figures of merits8,9&10 Figure Level Mass/charge ratio m/z 5-3000 (quadrupole), 5-12000 (TOF) Resolution 9000 FWHM at 800 m/z Mass accuracy 5 ppm between 150-900 m/z Two modes of MS MS and MS/MS Flow rate for nanoLC 150-300 nL/min Nanospray 20-40 nL/min Price range EUR 40,000 Software BioAnalyst version QS 2.0 L.O.D. ~ Femtomole, ~attomole in the presence of PITC protein derivatives The control and standard solutions for calibration will be made using hamster or mice brains infected with BSE specifically. Reference: QSTAR Tandem Hybrid System, http://www.tau.ac.il/lifesci/units/proteomics/qstar.html (accessed Dec 6, 2011)Summing up The nanoLC/MS/MS was considered to be the best technique due to high selectivity and it provides high sensitivity for qualitative measurements. MS/MS was used instead of LIF due to higher signal to noise ratio. Q-TOF MS/MS can detect the prions in biological matrices in sub-femtomole amounts. The sensitivity can be increased by the use of phenylisothiocyanate (PITC) protein derivatives. Bovine trypsin (or proteinase K) digests other proteins in the sample leaving only misfolded proteins as they are resistant to hydrolysis
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