Osman Adil Jamshed Chem th December, 2011 PrP C PrP Sc Reference: Prion structure, %20Structure.html (accessed Oct )

Prions Prions are misfolded form of proteins – beta sheets preferred over alpha helix - classified as infectious pathogens 1. Responsible for fatal neurodegenerative diseases in mammals by modifying the secondary structure of cellular prion protein (PrP c ) to infected scarpie prion protein (PrP Sc ) 1. Transmissible spongiform encephalopathies – the transmissible form of prion diseases – spongiform as it causes sponge like lesions in the brain 2. Creutzfeldt – Jakob disease (CJD) in humans, Bovine spongiform encephalopathy (BSE) in cattle, Scarpie of sheep 2. 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, (accessed Dec 7, 2011)

Analytical Problem Prions are extremely stable and resistant to chemical and physical agents like heat, acids, alkalis, detergents and enzymatic proteolysis 1&2. It is a “slow virus” that is fatal and virtually undetectable until its too late 2. Simple cooking of foodborne variant Creutzfeldt-Jakob disease infected meat products especially brain, does not stop the transmission 1. 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 –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 = PrP Sc ; 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. Studies

References : 3 Shkundina, I. S.; Ter-Avanesyan, M. D.; Biochemistry (Moscow), 2007, 72, 13, pp Onisko, B.; Dynin, I.; Requena, J.; Silva, C.; Erickson, M.; Carter, J.; J. Am. Soc. Mass Spectrom., 2007, 18, Ramsay, L.; Dickerson, J.; Dovichi, N.; Electrophoresis 2009, 30, 297–302

Diagram of Q-TOF mass spectrometer operating in MS (upper) and MS/MS mode (lower) modes. Reference: Dunn, M.; Industry assignment; (accessed Dec 6, 2011)

Homogenates will be centrifuged for 10 min at 5000 x g at 4 o C. 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 preparation 4 Supernatants of brain or nervous tissue homogenates were aliquoted and stored at -80 o C. Before analysis, bovine trysin (3.3μg in 33 μL water) digestion will be carried out at 37 o C overnight. The digestion will be stopped by adding formic acid (2μL) to give a pH of Onisko, B.; Dynin, I.; Requena, J.; Silva, C.; Erickson, M.; Carter, J.; J. Am. Soc. Mass Spectrom., 2007, 18,

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, a/detail.cda?id=11316&type=Education Feature&chId=10&page=1 (accessed Dec 6, 2011) a/detail.cda?id=11316&type=Education Feature&chId=10&page=1 Reference: Prions formation n%20Structure.html (accessed Oct )

Figures of merits 8,9&10 FigureLevel Mass/charge ratiom/z (quadrupole), (TOF) Resolution9000 FWHM at 800 m/z Mass accuracy5 ppm between m/z Two modes of MSMS and MS/MS Flow rate for nanoLC nL/min Nanospray20-40 nL/min Price rangeEUR 40,000 SoftwareBioAnalyst 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, (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 and cleavage. Using protein cyclic misfolding amplification 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…

References 1. Prions and Transmissible Spongiform Encephalopathies, FDA aturalToxins/BadBugBook/ucm htm (accessed Dec 6, 2011) aturalToxins/BadBugBook/ucm htm 2. Prusiner, S.; Proc. Natl. Acad. Sci.,1998, 95, pp – Shkundina, I. S.; Ter-Avanesyan, M. D.; Biochemistry (Moscow), 2007, 72, 13, pp Onisko, B.; Dynin, I.; Requena, J.; Silva, C.; Erickson, M.; Carter, J.; J. Am. Soc. Mass Spectrom., 2007, 18, Ramsay, L.; Dickerson, J.; Dovichi, N.; Electrophoresis 2009, 30, 297– Serbec, V.; Bresjanac, M.; Popovic, M.; et al, J. Bio. Chem. 2004, 279, 5, Hybrid quadrupole-TOF (Q-TOF) for MS/MS, 0&page=1 (accessed Dec 6, 2011) 0&page=1 8. Equipment, (accessed Dec 6, 2011) 9. QSTAR Tandem Hybrid System, (accessed Dec 6, 2011) 10. Dunn, M.; Industry assignment; (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