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Osman-Jamshed-Analytical-problem-Detecting prions by analytical methods.

Prions are misfolded form of proteins acting as an infectious agent, which are responsible for the transmissible spongiform encephalopathies in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as "mad cow disease") in cattle and Creutzfeldt-Jakob disease (CJD) in humans. These misfolded proteins affects the structure of the brain or the nervous system in a negative way, and is currently untreatable and fatal.
My hypothesis is that despite the aggressive regulations against the infected meat products in US, there are many countries that do not have these kind of regulation to protect public from prions, so detection of prions in such countries might help stop its spread.
This disease is transmitted by infectious meat and simple cooking does not affect these stable misfolded proteins. So the only way to avoid contracting this fatal disease is detection before its transmitted. However, detection of the infectious isoform is made difficult by the fact that it is quit similar to the normal cellular isoform of this protein.

For detecting this harmful protein few of the methods developed include FT-IR spectroscopy, nanoLC/MS/MS, and different electrophoretic methods. The analyte is usually in the blood, spinal fluids or tissue and/or bone fragments.

References:
Onisko, B.; Dynin, I.; Requena, J.; Silva, C.; Erickson, M.; and Cartera, J. J Am Soc Mass Spectrom 2007, 18, 1070-1079

Beekes, M.; Lasch, P.; Naumann,D. Veterinary Microbiology 123 (2007) 305-319

MOMCILOVIC, D.; RASOOLY, A.; J. Food Prot., Vol. 63, No. 11, 1602-1609

UV-vis absorption spectroscopy:
The prion, my analyte in blood or serum matrix, cannot be detected on it own by UV-vis spectrometry. However, isolated prions have shown high affinity for copper ions which can make the copper prion ligand complex detectable in UV-vis spectroscopy. The article I used had different concentration of copper ions and prions used. Like for Cu(II):Prion (L) = 1:1 the maximum wavelength was 600 nm, the molar absorptivity was 140, at pH 7.3, in MOPS (40 mM) solvent. For Cu : L = 2:1 the maximum wavelength was 598 nm, the molar absorptivity was 109, at pH 7.3, in MOPS (40 mM) solvent. There were two other concentrations 3:1 and 4:1 data given in the article as well. For the 3:1 concentration the maximum wavelength was 595 nm, the molar absorptivity was 103, at pH 7.3, in MOPS (40 mM) solvent. And for the 4:1 concentration the maximum wavelength was 592 nm, the molar absorptivity was 90, at pH 7.3, in MOPS (40 mM) solvent. The article also mentioned the binding site of the prions and how the nitrate group present can reduces the Cu(II) ion to Cu(I) leading to different species formed and precipitation observed. The article talks about isolated prions but i think the same can done for detecting prions in the blood or serum sample but the concentration ratio might be different which can then be adjusted to get usable data from UV-vis spectroscopy.
Reference:
Bonomo,R.; Natale, G.; Rizzarelli, E.; Tabbi, G.; Vagliasindi, L.; Dalton Trans., 2009, 2637-2646.

The analytical problem close to mine is Andrew Xayamongkhon as his matrix is also blood and it involves nitrates reacting with biological molecules present. His problem is similar to mine as we both are using the same matrix and in both our problem nitrate is reacting with the either the matrix (in his case) or copper ion (in my case) for the UV vis.
Another problem close to mine is Matt Marah - Analytical Problem: Cadmium Levels in Blood as we both are looking for harmful compounds in blood.

Similar Analytical Problems
The problems close to mine because of the same matrix blood include Nitric oxide and muscle growth (Andrew Xayamongkhon), Perfluorooctanoic acid levels in human blood (Andrew Szeliga), Cadmium Levels in Blood (Matt Marah), and DEHP Leaching from PVC into Contents of Medical Devices (Rajvi Mehta). The studies would be very different for each of these problems compared to mine and the only similarity that I think would be the blood matrix we all are using.
My problem is related to food industry and problems similar to mine according to relevance includes Nanoparticles Accumulate in the Food Chain (Nate Vetter) and I-131 in Japanese Milk Supply (Joe Zibley). The similarity in studies might include the fact that random sampling of the food products would have to be used at least for my problem and Joe's problem. The differences include the techniques used for detecting our analytes.

Hypothesis: Meat products, blood, tissues, organs, bone marrow, etc. can transmit prions, an untreatable and fatal disease, that is not only hard to detect due to very small amount of analyte present, but also unaffected by the usual methods used to cook contaminated meat or make it into meat products.

Studies: (A) Identify the regions where the prions were last officially reported, the regions most susceptible to prions outbreak - regions which use animal products in animal feed like European countries, the regions where infected products could have been shipped to unknowingly, the regions where there is no official regulations to prevent the spread of prions (developing nations like Pakistan). (B) Random sampling of the products in regions of interest and humans that report the symptoms associated with disease to detect the presence of prions. (C) Use different techniques to determine the most effective way to detect the mis-folded proteins in various matrixes (D) Investigate how far the transmission has spread over if the prions are detected in any region, by following the product supply chain and the human carriers.

The analyte concentration in a homogenate fraction is 2 micro grams per mL in a 150 mL solution. There are other concentrations of prions in different kinds of samples with the central nervous system having the highest concentration. But this concentration can be increased by different techniques before actually analyzing for prions.
Reference:
Gabizon, R., MCKINLEY, M.; Proc. Natl. Acad. Sci. USA, 85 ,1988, pp.6617-6621.

Blog 5
Prions on its own are not fluorescent but the fluorescent dye thioflavin T (ThT) can be used to detect the prion seeding of rPrPc polymerization. The (microtiter) Polarstar or Fluostar plate reader (320 individual wells) can be used to measure THT fluorescence in relative fluorescent units (rfu) (with seeding saturation occurring at ~260,000, in the reference article). The ThT fluorescence measurements are 450 +/- 10 nm excitation and 480 +/- 10 nm emission (bottom read, 20 flashes per well, manual gain of 2000, and 20 micro seconds integration time were used in the reference analysis method). If the concentration in the sample is supposed to be quite low, the concentration of prions can be increased using protein misfolding cyclic amplification (PMCA) to dectect as little as 1 ag of PrPsc .

Reference:
Wilham, J.; Orru, C.; Bessen, R.; Atarashi, R.; Sano, K.; Race, B.; Meade-White, K.; Taubner, L.; Timmes, A.; Caughey, B.; PLoS Pathogens, Vol. 6, Issue 12, 2010, pg 1-15.

Blog 6
Prions can be formed when the PrP 33-35 is replaced by PrP 27-30, it changes PrPc to PrPsc, the common protein to the infectious protein.
View image

(Reference for photo 1: http://www.atsu.edu/faculty/chamberlain/Website/Lects/Prions.htm (accessed Oct 25 2011))

This causes a conformational change in the PrP protein from an a-helix to a b-sheet dominant structure. Structure 'a' is normal PrP with only alpha helix and structure 'b' is prion with beta sheets.

View image

(Reference for photo 2: http://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%2010/Prion%20Structure.html (accessed Oct 25 2011))

According to some sources the normal proteins also have beta sheets but the number of beta sheets is more in the prions.
View image

(Reference for photo 3:
http://www.scq.ubc.ca/prions-infectious-proteins-repsonsible-for-mad-cow-disease/
Art by Jen Philpot (accessed Oct 25 2011))
The structure of prions has not been completely identified but biologist unanimously agree that the misfolding of the protein involves alpha helix to be be replaced by beta sheets in the structure of prions.

The google search for prions standard sample did not yield any results. So for calibration the standards have to be made. This can be done using mice that are injected with 30 μl, for instance, of infected brain extract inserted into the right parietal lobe. The inocula of the mice can be of any one of the different strains of prions. The incubation time will vary depending on the strain used. The brains of these mice or its portions will be extracted and homogenized to be used as the standard. The homogenate can be prepared in phosphate buffered saline lacking calcium and magnesium ions using the infected brain tissue. The tissue will be initially dissociated using a sterile disposable homogenizer, and the suspension will be subjected to repeated extrusion through different gauge syringe needles (18 gauge and then 22 gauge). The solutions can be assayed for the desired properties, e.g. PrPsc concentration and overall prion concentration, diluted 10-fold and stored as the standards.

Reference:
Stanley B.; "Prion protein standard and method of making the same", osdir.com, Patents archive.
http://osdir.com/patents/Chemistry-resins/Prion-protein-standard-method-making-06962975.html (accessed Oct 25 2011)


Blog 7
No, prions cannot be analyzed or quantified by atomic spectrometries directly. Prions can bind with copper so atomic absorption spectroscopy can used to show a reduction in the copper concentration and the prions can be quantified indirectly in this way. The analyte being measured is copper ions (the bound Cu(II) in the protein metal mix); absorption at 325.2 nm; the sample ashed using plasma processor TePLa 100-E (Technics Plasma), absorbed in nitric acid, and analyzed by a Zeeman 3030 (Perkin Elmer) flameless atomic absorption spectrophotometer after rapid atomization at 2000 oC in a graphite-tube cuvette HGA-70 (Perkin Elmer). Atomic absorption spectrometry was used as the concentration of Cu(II) ions is being determined in a protein-metal ion mixture and compared to the blank copper ions solution to calculate the difference in concentrations , which using absorption and Beer's law can easily be achieved.

Reference: Brown, D.; Qin, K.; Herms, J.; Madlung, A.; Manson, J.; Strome, R.; Fraser, P.; Kruck, T.; von Bohlen, A.; Schulz-Schaeffer, W.; Giese, A.; Westaway, D.; Kretzschmar, H.; Letters to Nature, Vol. 390, 1997, pp 684-687.

Blog 9: Chromatographic techniques
For separating prions gas , reverse phase, HILIC, ion-exchange, size-exclusion and affinity chromatography could be used. All these techniques are suitable for separating prions as the prions are very stable, hydrophobic and large polar molecules. Proteins are chiral too but the size and the numerous chiral centers present makes the it very difficult for prions to be separated by chiral chromatography.
Reverse phase liquid chromatography will be my first choice as it is most widely available and used chromatography technique. The availability will be useful when samples from different place can be analyzed at different labs after calibrating the equipment making the process faster.
A possible column would be AQ-C18 by SiliaChrom, with hiqh purity spherical silica, 5 micrometer particle size, 250 mm column length, 20 mm internal diameter of the column, pH stability 1.5-9.0 (possibly). The catalogue number is H151805E-Y250 and the company name is Silicycle.
The mobile phase will be 80% MeOH and 20% phosphate buffer pH 7.0.
I will use MS/MS (quadrapole/time of flight) as I expect very low levels (less than nano molar) of prions in sample if any present. This concentration can be increased using hte PMCA (protein misfolding cyclic amplification) technique but if analyzing without the amplification of prions MS/MS seems to be a good as it can detect very quantities of prions, if present.
References:
Silicylce, http://www.silicycle.com/applications/analysis/hplc-columns/siliachrom-aq-c18-universal-hplc-column/h1518-p
Onisko, B.; Dynin, I.; Requena, J.; Silva, C.; Erickson, M.; Carter, J.; J. Am. Soc. Mass Spectrom., 2007, 18, 1070-1079.
Reverse phase column, http://www.gc-lc.com/column_RP.htm

Blog 10
My preferred technique would be nanoLC-MS/MS as it has shown ability to detect attomole quantities of prions. No one is using exactly the same technique for separation and detection but the problems with very similar techniques include "Perfluorooctanoic acid levels in human blood" (prefers LC-MS/MS); "Triclocaban in Human Urine", "Brevetoxin levels in Ocean", and "Pesticides and Toxins in fragrances and natural flavors"(all 3 prefers RPLC-MS/MS). These four prefers MS/MS but the separation techniques are a little different although they all are types of chromatography.
(I posted it as a comment first but it is still spamming my comments so I am posting in the entry too)


Blog 11
All the types of CE (CZE, cIEF and CGE) except for the MEKC would be suitable for separating prions from the normal proteins, and tissue, blood and bone matrixes. MEKC depends on the hydrophobicity of a molecule but the there is no known distinction in terms of sequence for hydrophobicity between prions and normal proteins yet (1). Without known difference in hydrophobicity MEKC cannot be used for separation and I was unable to find any paper using this technique for separation of prions. My first choice would be cIEF as the normal proteins have a higher isoelectric point (pI around 6.5) and the prions have a lower isoelectric point (pI around 4). This difference in the polarity allows for separation of prions even if the amount is in attomole. cIEF focusing will be performed on a Beckman P/ ACE 5500 (Beckman Instruments) controlled by P/ACE station software (Beck- man Instruments). A cIEF 3-10 Kit (Beckman Instruments) contained a neutral capillary 45 cm by 50 μm, ampholytes ranging from 3-10, and cIEF gel will be used. Markers that will be used are RNAase (pI of 9.45), carbonic anhydrase (pI of 5.98), b-lactoglobulin (pI of 5.10) and CCK flanking peptide (pI of 2.75). The catholyte solution will be 20 mM NaOH and the anolyte will be 91 mM phosphoric acid in the cIEF gel. The sample will be prepared using 4 μl of the ampholyte solution mixed with the cIEF gel and added to 5 μl of sample or makers mixed well and centrifuged at 7000 g to remove bubbles. Using a high- pressure injection for 1 min the capillary will be filled. The proteins will be focused for 2 min at a voltage of 13.5 kV. A low-pressure rinse will be applied simultaneously with the field strength of the electric field being maintained at 500 V/cm, to mobilize the focused proteins (2). The detector I would use is MS/MS as this detector can detect prions quantities as low as attomoles (3).
References:
1. "Protein Conformation and the Concept of Misfolding", http://www.nature.com/scitable/topicpage/protein-misfolding-and-degenerative-diseases-14434929
2. Schmerr, M.; Cutlip, R.; Jenny, A. Journal of Chromatography A, 1998, 802, pg 135-141.
3. Onisko, B.; Dynin, I.; Requena, J.; Silva, C.; Erickson, M.; Carter, J.; J. Am. Soc. Mass Spectrom., 2007, 18, 1070-1079.

Blog 13
Prions are not electroactive as they cannot be oxidized or reduced in a quantitative manner. The prions selectively binds to copper (II) ions. This can used to quantify prions by measuring the change in absorbance by UV-vis spectroscopy with excitation wavelength at 280 nm . The beers law will be used to convert absorbance to concentration of the unbound copper ions. First get the metal free prions and determine the concentration. React the prions with cuprizone, a Cu (II) ion chelating reagent and determine the change in concentration of the cupric ions. Using this data make a calibration curve and then the curve to quantify the prions in a sample with a ion-selective.
Reference:
Stockel, J.; Safar, J.; Wallace, A.; Cohen, F.; Prusiner, S.; Biochemistry 1998, 37, 7185-7193.

Comments

cuprizone has copper ions and can interact with the prions. UV vis was used to make the calibration curve of how the concentration of the ions change by reacting with the prions. So when the ion selective electrode is used the change in the copper ion concentration would the give the concentration of prions in the solution.

Blog 13. It is unclear why you have to use cuprizone or a UV detector. How do these components play a role in the creation of an ion selective electrode? - 0. 5 pt.

Blog 9. Good answers. GC does not work for proteins because they decompose before they become volatile.
Blogs 9 and 10. Good answers.

Blog 10
My preferred technique would be nanoLC-MS/MS as it has shown ability to detect attomole quantities of prions. No one is using exactly the same technique for separation and detection but the problems with very similar techniques include "Perfluorooctanoic acid levels in human blood" (prefers LC-MS/MS); "Triclocaban in Human Urine", "Brevetoxin levels in Ocean", and "Pesticides and Toxins in fragrances and natural flavors"(all 3 prefers RPLC-MS/MS). These four prefers MS/MS bu the separation techniques are a little different although they all are types of chromatography.

Where is your analyte? The supernatant, the sucrose layer, or the pellets? When the storage period is over, what do you do with it? (-0.5 pt)

Blog 8
Homogenation: Scrapie-infected tissue samples will be homogenized in 10 volumes (wt/vol) PBS (sodium perborate) with a Kendall (Mansfield, MA) disposable plastic homogenizer.
Centrifugation: The supernatant will be separated from the homogenized mixture of the sample centrifuging at 4°C for 1 h at 100,000 g.
Extraction: Supernatant will be layered on to a 20% (w/v) sucrose cushion and clarified by ultracentrifugation at 160 000 g for 4 h.
Drying and storage: The pellets, the sucrose layer and the remaining supernatant will be stored at −80◦C.

References:
Deleault, N.; Harris, B.; Rees, J.; Supattapone, S.; PNAS, 2007, 104, 23, 9741-9746.

Muller, H.; Strom, A.; Hunsmann, G.; Stuke, A.; Biochem. J., 2005, 388, 371-378.

Blog 6. There are abbreviations, not chemical information. What are the amino acids in the Prp 35-37 and 27-30 sequences? (-0.25 pt).
The purification procedure seems convoluted. If you were going to analyze only a small part of the protein, synthetic peptides may be used. We can talk about it, if you want.

Blog 7. Detection of Cu by AAS is useful to observe variations in copper levels, but such variations will result from interactions with many biomolecules, not only prions.The method will be unselective. Second part of question 3? MS analysis (-0.5 pt).


Blog 7
No, prions cannot be analyzed or quantified by atomic spectrometries directly. Prions can bind with copper so atomic absorption spectroscopy can used to show a reduction in the copper concentration and the prions can be quantified indirectly in this way. The analyte being measured is copper ions (the bound Cu(II) in the protein metal mix); absorption at 325.2 nm; the sample ashed using plasma processor TePLa 100-E (Technics Plasma), absorbed in nitric acid, and analyzed by a Zeeman 3030 (Perkin Elmer) flameless atomic absorption spectrophotometer after rapid atomization at 2000 oC in a graphite-tube cuvette HGA-70 (Perkin Elmer). Atomic absorption spectrometry was used as the concentration of Cu(II) ions is being determined in a protein-metal ion mixture and compared to the blank copper ions solution to calculate the difference in concentrations , which using absorption and Beer's law can easily be achieved.

Reference: Brown, D.; Qin, K.; Herms, J.; Madlung, A.; Manson, J.; Strome, R.; Fraser, P.; Kruck, T.; von Bohlen, A.; Schulz-Schaeffer, W.; Giese, A.; Westaway, D.; Kretzschmar, H.; Letters to Nature, Vol. 390, 1997, pp 684-687.

BLOG 5 - Good answers!

BLOG 4. I do not think that the proposed studies will help you test your current hypothesis. You seem to have two hypotheses: (1) hard to detect in meat products, (2) inneffective methods to eliminate prions from meat products. The first study (A), only define geographic conditions. The second study (B) may be relevant but random sampling will not work if the incidence of the disease is low. (C) Use of techniques is not a study. (D) Spread of the disease does not help either. Make an office hour appointment to talk about studies (-1 pt).

Blog 5
Prions on its own are not fluorescent but the fluorescent dye thioflavin T (ThT) can be used to detect the prion seeding of rPrPc polymerization. The (microtiter) Polarstar or Fluostar plate reader (320 individual wells) can be used to measure THT fluorescence in relative fluorescent units (rfu) (with seeding saturation occurring at ~260,000, in the reference article). The ThT fluorescence measurements are 450 +/- 10 nm excitation and 480 +/- 10 nm emission (bottom read, 20 flashes per well, manual gain of 2000, and 20 micro seconds integration time were used in the reference analysis method). If the concentration in the sample is supposed to be quite low, the concentration of prions can be increased using protein misfolding cyclic amplification (PMCA) to dectect as little as 1 ag of PrPsc .

Reference:
Wilham, J.; Orru, C.; Bessen, R.; Atarashi, R.; Sano, K.; Race, B.; Meade-White, K.; Taubner, L.; Timmes, A.; Caughey, B.; PLoS Pathogens, Vol. 6, Issue 12, 2010.

Hypothesis: Meat products, blood, tissues, organs, bone marrow, etc. can transmit prions, an untreatable and fatal disease, that is not only hard to detect due to very small amount of analyte present, but also unaffected by the usual methods used to cook contaminated meat or make it into meat products.

Studies: (A) Identify the regions where the prions were last officially reported, the regions most susceptible to prions outbreak - regions which use animal products in animal feed like European countries, the regions where infected products could have been shipped to unknowingly, the regions where there is no official regulations to prevent the spread of prions (developing nations like Pakistan). (B) Random sampling of the products in regions of interest and humans that report the symptoms associated with disease to detect the presence of prions. (C) Use different techniques to determine the most effective way to detect the mis-folded proteins in various matrixes (D) Investigate how far the transmission has spread over if the prions are detected in any region, by following the product supply chain and the human carriers.

The analyte concentration in a homogenate fraction is 2 micro grams per mL in a 150 mL solution. There are other concentrations of prions in different kinds of samples with the central nervous system having the highest concentration. But this concentration can be increased by different techniques before actually analyzing for prions.
Reference:
Gabizon, R., MCKINLEY, M.; Proc. Natl. Acad. Sci. USA, 85 ,1988, pp.6617-6621.

BLOG3.
- (a) There is a problem that looks and proteins and allergens in foods.
-(b) The studies used to solve this problem will likely use similar extraction procedures isolate and determine quantitities of low abundance proteins (-0.25 pt).
-(c) So what studies will be different? (-0.25 pt).

The topic is ok, but you are not defining a hypothesis. What do you think is happening? What is the possible explanation? Answers to questions like these ones, can help you define your hypothesis. You may want to drop by office hours to talk in person.

Hello Osman. My name is Andrew and my analyte of interest is also found in the blood. My analytical problem is to determine the amount of nitric oxide is in the blood sample. Because the nitric oxide has a short half life and reacts quickly with biological molecules to create, for example, NO3- and NO2-, I choose to test the nitrate/nitrite ratio in the blood to give me an indication of NO. How do you plan to isolate the protein from the blood? For my analytical problem, I need to isolate plasma nitrate/nitrite. Do you have any suggestions?

I did mention in the second paragraph that I want to test meat products for prions or do i need to change my hypothesis.

I want to test the livestock or meat products for prions before its been sold or used in order to make sure that this disease is not transmitted to other animals or humans who eat them.

Good description of the problem and its importance. What is the hypothesis that you plan to test? (-0.2 pts)