1 / 12

Detection of prions by analytical methods.

Detection of prions by analytical methods. Osman Adil Jamshed Chem 4101 9th December, 2011. Reference: Prion structure, http://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%2010/Prion%20Structure.html (accessed Oct 25 2011) . PrP C. PrP Sc. Prions.

barton
Download Presentation

Detection of prions by analytical methods.

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Detection of prions by analytical methods. Osman Adil Jamshed Chem 4101 9th December, 2011 Reference: Prionstructure, http://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%2010/Prion%20Structure.html (accessed Oct 25 2011) PrPC PrPSc

  2. 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.

  3. 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)

  4. 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.

  5. Hypothesis • 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. • 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 enforcedby factories in the region. • Analyte = PrPSc; Matrices = biological, brain and CNS tissue. Studies

  6. References : 3Shkundina, 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, 297–302

  7. 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)

  8. Sample preparation4 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. Homogenates will be centrifuged for 10 min at 5000 x g at 4 oC. 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.

  9. NanoLC/ESI/Q-TOF MS/MS Q-TOF MS/MS Figure Applied Biosystems MDS SCIEX QStar Pulsar equipped with ProxeonBiosystemsnanoelectrospray source was used for detection and general ESI process (image on the far right). 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) ESI process Figure Reference: Prions formation http://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%2010/Prion%20Structure.html (accessed Oct 25 2011)

  10. Figures of merits8,9&10 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)

  11. 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/MScan 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 and cleavage. • Using protein cyclic misfoldingamplification the concentration in a sample can be increased and used as a standard. • Essentially the reason behind detection of prions is that prevention is better than cure…

  12. References • Prions and Transmissible Spongiform Encephalopathies, FDAhttp://www.fda.gov/Food/FoodSafety/FoodborneIllness/FoodborneIllnessFoodbornePathogensNaturalToxins/BadBugBook/ucm071397.htm (accessed Dec 6, 2011) • Prusiner, S.; Proc. Natl. Acad. Sci.,1998, 95, pp. 13363–13383 • Shkundina, I. S.; Ter-Avanesyan, M. D.; Biochemistry (Moscow), 2007, 72, 13, pp. 1519-1536 • Onisko, B.; Dynin, I.; Requena, J.; Silva, C.; Erickson, M.; Carter, J.; J. Am. Soc. Mass Spectrom., 2007, 18, 1070-1079. • Ramsay, L.; Dickerson, J.; Dovichi, N.; Electrophoresis 2009, 30, 297–302 • Serbec, V.; Bresjanac, M.; Popovic, M.; et al, J. Bio. Chem. 2004, 279, 5, 3694-3698. • 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) • Equipment, http://www.unige.ch/sciences/sms/2.html, (accessed Dec 6, 2011) • QSTAR Tandem Hybrid System, http://www.tau.ac.il/lifesci/units/proteomics/qstar.html (accessed Dec 6, 2011) • Dunn, M.; Industry assignment; http://www.colorado.edu/chemistry/chem5181/QSTAR%20Pulsar.pdf, (accessed Dec 6, 2011) • Wilham, J.; Orru, C.; Bessen, R.; Atarashi, R.; Sano, K.; Race, B.; Meade-White, K.; Taubner, L.; Timmes, A.; Caughey, B.; PLoS Pathogens, 2010, 6, 12, pg 1-15

More Related