Quantitating PrP Polymorphisms Present in Prions from Heterozygous

Subscriber access provided by GAZI UNIV

Article

Quantitating PrP polymorphisms present in prions from heterozygous scrapie-infected sheep. Christopher J. Silva, Melissa L. Erickson-Beltran, Colleen Hui, Juan José Badiola, Eric M Nicholson, Jesus Rodriguez Requena, and Rosa Bolea Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03822 • Publication Date (Web): 11 Dec 2016 Downloaded from http://pubs.acs.org on December 20, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Analytical Chemistry

100

171 Q

171 Q

171 Q

171 Q

136 V

136 V

136 V

136 V

171 H

171 H

171 H

171 H

136 A

136 A

136 A

136 A

50

0

+PK -PK -PK +PK -PK -PK +PK +PK 171H/Q (2) 136A/V (1) 136A/V (2) 171H/Q (1) 1 2 3 4 5 6 7 8 Percentage of each polymorphism found in prions from heterozygous sheep

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Title: Quantitating PrP polymorphisms present in prions from heterozygous scrapieinfected sheep. By: Christopher J. Silva1*, Melissa L. Erickson-Beltran1, Colleen Hui1,2, Juan José Badiola3, Eric M. Nicholson4, Jesús R. Requena5, Rosa Bolea3

1. Produce Safety & Microbiology Research Unit, Western Regional Research Center, United States Department of Agriculture, Agricultural Research Service, Albany, California 94710, United States of America. 2. Current address: Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, United States of America. 3. Veterinary Faculty, Centro de Investigación en Encefalopatías y Enfermedades Transmisibles Emergentes (CIEETE), Universidad de Zaragoza, 50013, Zaragoza, Spain. 4. Virus and Prion Research Unit, National Animal Disease Center, United States Department of Agriculture, Agricultural Research Service, Ames, Iowa 50010, United States of America. 5. CIMUS Biomedical Research Institute, University of Santiago de CompostelaIDIS, Santiago de Compostela, Spain. * Correspondence to: Christopher J. Silva; USDA, ARS, WRRC 800 Buchanan Street Albany California 94710, USA. Phone 510.559.6135. FAX 510.559.6429. email:[email protected]. Abstract Scrapie is a prion (PrPSc) disease of sheep. The incubation period of sheep scrapie is strongly influenced by polymorphisms at positions 136, 154, and 171 of a sheep’s normal cellular prion protein (PrPC). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic peptides [M132LGSXMSRPL141 (X = A or V), Y153XENMY158 (X= H or R), and Y166RPVDXY172 (X = H, K, Q, or R)] that could be used to detect and quantitate polymorphisms at positions 136, 154, and 171 of sheep PrPC or PrPSc. These peptides were used to develop a multiple reaction monitoring method (MRM) to detect the amounts of a particular polymorphism in a sample of PrPSc isolated from sheep heterozygous for their PrPC proteins. The limit of detection for these peptides was less than 50 attomole. Spinal cord tissue from heterozygous (ARQ/VRQ or ARH/ARQ) scrapie-infected Rasa Aragonesa sheep was analyzed using this MRM method. Both sets of heterozygotes show the presence of both polymorphisms in PrPSc. This was true for samples containing both proteinase K (PK)-sensitive and PK-resistant PrPSc and samples containing only the PK-resistant PrPSc. These results show that heterozygous animals contain PrPSc that is composed of significant amounts of both PrP polymorphisms.

1


Page 2 of 20

Page 3 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Scrapie is a prion disease of sheep and goats.1 Prion diseases are characterized by a long incubation period followed by the comparatively brief appearance of symptoms and death.2 Prions (PrPSc) are able to convert a normal cellular prion protein (PrPC) into a prion and thereby propagate an infection. The incubation can be extended if the protein sequence of PrPC is different from that of PrPSc. This extension of the incubation period has historically been referred to as a “species barrier”, since it was based on the observation that the incubation of a prion disease was extended when it was transmitted to a new species.3 If members of the same species have polymorphisms in their PrPC protein, then a “species barrier” may also be observed, even though they are of the same species. Sheep possess PrPC polymorphisms at positions 136, 154, and 171 that strongly influence their susceptibility to prion disease.4-9 This can result in dramatically different incubation periods for sheep with different PrPC polymorphisms that are experimentally infected with the same amount of prions.10 Among sheep naturally infected with classical scrapie, valine (V) at position 136 is associated with a greater susceptibility to the disease, while alanine (A) is associated with a lower incidence of scrapie.4, 8, 11, 12 Sheep heterozygous for A and V at position 136 are also vulnerable to classical scrapie.5, 10, 13 Histidine (H) at position 154 confers some resistance to classical scrapie in sheep, but, in transgenic mouse models, seems to facilitate the transmission of atypical scrapie and BSE.14 Histidine at position 154 is associated with atypical scrapie.15-18 Sheep with glutamine (Q) at position 171 are very susceptible to classical scrapie and those with H at position 171 appear to be neutral, while lysine (K) at position 171 imparts resistance and arginine (R) at the same position confers near complete resistance to classical scrapie.4, 5, 14, 19 The role that heterozygotes play in bridging the species barrier is unresolved. Unfortunately there are few methods of detecting and quantitating the polymorphisms that comprise the PrPSc in PrP heterozygotes. Antibodies have been used to distinguish between the components of the PrPres present in a heterozygous human patient.20 The results of this study indicated that only one of the polymorphisms was found in the PrPres.20 Other researchers showed that the PrPC present in a heterozygous host was evenly divided between the two polymorphisms.21, 22 An antibody has been used to identify sheep polymorphisms containing an R at position 171.23 Another antibody-based approach has been used to detect the amount of PrPSc containing R and Q at position 171.24, 25 Unfortunately antibody-based approaches are limited by the availability of the requisite antibodies. Mass spectrometry was used to detect the relative amount of each polymorphism present in human and sheep PrPSc samples.22, 26 While that mass spectrometry-based approach is not limited by the availability of antibodies, it is reliant on the proteolytic enzyme trypsin. Both PrPC and PrPSc have covalent post translational modifications which complicate structural analysis.27, 28 Both possess similarly varying patterns of glycosylation at either or both of two asparagine residues in the protein.29-31 Both also have a glycophosphatidylinositol (GPI) anchor that is similarly varied in its glycosylation and an identical single disulfide bond.32-34 PrPC and PrPSc are isosequential, having the same amino acid sequence.28 The only demonstrated structural difference between PrPC and PrPSc is conformational.2 PrPC is monomeric, while PrPSc is a multimer that

2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

stabilizes its conformation.35 The multimers of PrPSc are composed of a fraction that is resistant to proteinase K (PK) and one that is PK sensitive.36-38 Analysis of the PrPSc protein is greatly facilitated by the enzymatic digestion of the denatured and disaggregated protein.39 After inactivation (denaturation), prions have been analyzed by a number of mass spectrometry-based techniques.39 The whole protein from denatured GPI-anchorless PrPSc has been analyzed by mass spectrometry.40 Unfortunately whole protein analysis is only applicable to GPI anchorless PrPSc, since it lacks both significant asparagine glycosylation and a GPI anchor.41, 42 Trypsin and LysC were used to digest denatured PrPSc to yield a set of characteristic tryptic peptides.28 Mass spectrometry based-analysis of these peptides showed that there were no covalent differences between PrPC and PrPSc.28, 43 Both tryptic and chymotryptic digestion of denatured PrPSc have been used to identify peptides that can be used to quantitate the amount of PrPSc in a sample using the very sensitive multiple reaction monitoring (MRM) method.44-47 Trypsin has been used to digest PK treated and non-PK treated denatured PrPSc in order to distinguish among prion strains.48 Trypsin and chymotrypsin were also used to digest PrPSc that was reacted with small molecule reagents in order to quantify the extent of the reaction of those reagents with the lysines in PrPSc.49 In practice mass spectrometry-based analysis of a proteolytic digestion of denatured Sc PrP has been used to quantitate the relative amount of PrP polymorphisms present in PrPSc.22, 26 Sheep PrP has approximately 22 lysines or arginines, depending on the specific polymorphic variant. Unfortunately, they are not evenly distributed, so the tryptic peptides around positions 136 and 171 are very large and difficult to analyze in solution owing to their poor solubility.22 As a result, these peptides are analyzed after an in-gel digestion.22, 26 Since SDS-PAGE oxidizes methionines, the methionines from ingel digested peptides are more extensively oxidized when compared to the same peptides derived from an in-solution digestion using the same enzyme.50, 51 In addition sheep PrP contains two arginine-proline bonds (139/140 and 167/168) in the 136 and 171 region, where cleavage is inherently variable and would complicate a trypsin-based analysis. The relative abundance of lysine and arginine near the 154 polymorphism means that the tryptic peptides containing 154 are small and difficult to analyze. Chymotrypsin has been used to digest PrPSc in order to study regions of the protein that are not suitable for These results suggest that MRM-based analysis when digested with trypsin.49 chymotryptic digestion is a useful means of obtaining results that trypsin digestion can not accomplish. We have developed a mass spectrometry-based MRM method of detecting and quantitating the PrP polymorphisms present in a sample of PrPSc from a heterozygous prion infected animal. We have applied this method to a set of naturally infected sheep. The results are reported here. Experimental Procedures (Materials and Methods) Chemicals. The sheep peptides (M132LGSAMSRPL141, M132LGSVMSRPL141, R159YPNQVY165, Y153HENMY158, Y153RENMY158, Y166RPVDHY172, Y166RPVDKY172, Y166RPVDQY172, and Y166RPVDRY172) were obtained from Elim Biopharmaceuticals (Hayward, CA) or Peptide 2.0 (Chantilly, VA). Throughout the manuscript, the

3


Page 4 of 20

Page 5 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


polymorphisms (variations in the amino acids at positions 136, 154, or 171) are underlined and in bold for the sake of clarity. The chemical composition of all of these synthetic peptides was confirmed by mass spectrometry. A greater than 95% chemical purity was demonstrated by LC/UV. The 15N-labeled internal standards were obtained by digesting the corresponding 15 N-labeled recombinant PrP protein with chymotrypsin. The structure of the relevant PrP proteins was determined by the genetic sequence of the clone used to express it. The sequence was further confirmed by mass spectrometry. The incorporation of 15N label into the PrP samples was estimated to be 99.8%. Preparation of the sheep recombinant PrP polymorphisms. Site directed mutagenesis was used to prepare genes that would express sheep PrP polymorphisms at positions 136 (A and V) 154 (H and R) and 171 (H, K, Q, and R) using standard molecular biology techniques (Supporting Information). Preparation of the 15N-labeled internal standards. The seven sheep genes were each cloned into BL21 cells. The clones were separately grown in minimal medium supplemented with 15NH4Cl and induced to express their respective PrP polymorphism (Supporting Information). Quantitative Mass Spectrometry: Nanospray LCMSMS. An Applied Biosystems (ABI/MDS Sciex, Toronto, ON) model 4000 Q-Trap instrument equipped with a nanoelectrospray source was used to perform nanospray liquid chromatography and tandem mass spectroscopy (LC-MS/MS). An aliquot (6 µL) of each digest was loaded onto a C-18 trap cartridge [Acclaim PepMap100, 5 µm, 100Å, 300 µm (inside diameter) x 5 mm (Dionex, Sunnyvale, CA)]. Salts were washed from the cartridge with an acetic acid/acetonitrile/heptafluorobutyric acid/water solution (0.5/1/0.02/99). The now salt-free bound peptides were eluted onto a reversed-phase column [Vydac (Hesperia, CA) 238EV5.07515, 75 µm x 150 mm]. The solvents were delivered with an Applied Biosystems model Tempo nanoflow LC system (ABI/MDS Sciex) with an autosampler, a column switching device, and a nanoflow solvent delivery system. Samples were eluted from the column with a binary gradient (A, 0.5% acetic acid in water, and B, 80% acetonitrile with 0.5% acetic acid). The flow rate was 250 nL/min with a 16 min linear gradient starting with 5% B and ending with 100% B. Elution with 100% B was conducted for 7 min followed by a return to 5% B over 4 min. The eluted samples were sprayed with a non-coated spray tip (FS360-20-10-N-20-C12, New Objective Inc., Woburn, MA) onto the Applied Biosystems source, Model Nanospray II. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode, alternating between detection of the analyte peptides and their appropriate 15Nlabeled internal standards. The mass settings for the chymotryptic peptides were empirically determined and may be found in the Supporting Information. The mass settings for the quantification were done with the IntelliQuant quantification algorithm using Analyst 1.5 software. Safety considerations Hazardous material, such as acetonitrile, was manipulated in a dedicated chemical safety hood. Scrapie is infectious. A dedicated biosafety level 2 (BSL2) laboratory was where all scrapie-containing samples were manipulated (Supporting Information).

4



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Results and Discussion Selection of suitable analyte peptides. The sheep PrP (Figure 1) sequences corresponding to the ARQ (A at position 136, R at position 154, and Q at position 171), VRQ, VHQ, AHQ, ARH, ARK, and ARR were digested in silico, using the ExPASy portal (http://www.expasy.org/tools/). In silico digestion with trypsin yielded one peptide suitable for detecting and distinguishing among the sheep polymorphisms at position 136. Its analog had been previously used for analyzing human polymorphisms.26 A set of three peptide analogs was identified when the sheep PrP polymorphisms were digested (in silico) with chymotrypsin, including M132LGSXMSRPL141 (X = A or V), Y153XENMY158 (X= H or R), and Y166RPVDXY172 (X = H, K, Q, or R) which correspond to polymorphisms at position 136, 154, and 171. These peptides were suitable for a multiple reaction monitoring (MRM)-based analysis. Based on this analysis, chymotrypsin was selected to digest the recombinant PrP polymorphisms.

H G W Q P G G G P G G G H G G G G G G R P Q G G G H G W Q S H G W Q P G G W Q G N Y P G G W Q P G G G G H L G V G A A A H M T P S K W Q P R G T W G P P R S Q V A A A G V K N K K P N G G Y S G N G G K K G Y L S M R L H G K 25 N H2 G M G A S P I 232 A F N O D V P Y V 136 R G N Y D 171 Q R Q Y Q P Y NH R Y O E Y S Y P O A Q Y R D N Y Y R OH Q S N M E R E Q Y F N 154 N V HO P O O P OH Q H T D O O I C sC V H2N M s N Q OO E V T I O V K R V Octarepeat GPI E M H Q T β-Sheet V anchor K I T I T T α-Helix D Sugar residue E T T T G K F N E Sugar S

P

antennae

Figure 1. Cartoon of sheep PrPC (ARQ). The location of the relevant amino acids and secondary structures are noted.52 GPI anchor and sugar antennae are based on the hamster and mouse structures. (Adapted from Dumpitak.53).

5


Page 6 of 20

Page 7 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Previously prepared samples of sheep (ARR) recombinant PrP (rPrP) were used to determine if the in silico results could be replicated in vitro. The sheep rPrP was digested with chymotrypsin. After digesting sheep rPrP (ARR) with chymotrypsin, the peptide digests were analyzed by qualitative mass spectrometry (Supporting Information). This analysis revealed that the peptides M132LGSAMSRPL141, Y153RENMY158, and Y166RPVDRY172 were present in the samples. This result indicated that chymotryptic digestion of sheep rPrP (ARR) yields the peptide predicted by the in silico analysis. Preparing the sheep rPrP polymorphisms. Six polymorphisms of the sheep Prnp gene were prepared. The Prnp gene used in these experiments expressed the mature PrP protein (25-233), lacking the leading sequence and the tailing sequence which are cleaved during the processing of the protein. The starting Prnp gene for sheep encoded the ARR polymorphism. This gene was modified by site directed mutagenesis to generate other sheep Prnp genes, encoding other sheep PrP polymorphisms (VRQ, VHQ, AHQ, ARH, ARK, and ARQ) (Supporting Information). The resulting genes were cloned into a pET-11a vector. The Prnp genes were sequenced to verify that the site directed mutagenesis was successful. These clones (VRQ, VHQ, AHQ, ARH, ARK, and ARQ) were used to prepare the required sheep rPrP. The plasmids were cloned into an E. coli cell line engineered to express high levels of recombinant protein (BL21). These cells were grown in minimal medium, which was supplemented with either 15NH4Cl or natural abundance NH4Cl. The clones were induced to overexpress their respective PrP polymorphisms. The resulting PrP proteins were isolated as inclusion bodies and then purified by immobilized metal chromatography (IMAC). They were obtained in high purity (> 90%). Analysis of the 15 N-labeled proteins revealed that they had a very high isotopic abundance (> 99.7%). The purified proteins were used in subsequent analyses. The 15N-labeled proteins were used to prepare internal standards. The unlabeled proteins were used to verify the in silico predictions. Analyzing sheep rPrP polymorphisms. Recombinant PrP from six sheep polymorphisms (VRQ, VHQ, AHQ, ARH, ARK, and ARQ) were individually digested with chymotrypsin. The resulting digests were individually analyzed by qualitative mass spectrometry. These data revealed the presence of M132LGSVMSRPL141, Y153HENMY158, Y153RENMY158, Y166RPVDHY172, Y166RPVDKY172, and Y166RPVDQY172 in the samples. In addition to these peptides, chymotryptic digestion also yielded a peptide (G94QGGSHSQW102) analogous to one containing important polymorphisms in deer PrP (G94QGGTHSQW102). This confirmed the predictions of the previous in silico analysis. These results indicated that chymotryptic digestion of sheep PrP polymorphisms will yield peptides suitable for a MRM-based analysis. Chymotryptic digestion of sheep PrP will yield a set of peptides that can be used to detect and quantitate the amount of PrP polymorphisms at position 136, 154, and 171 in a sample containing two PrP proteins. Optimizing the chymotryptic peptides for MRM analysis. Chemically synthesized peptides (> 95% purity) were used to optimize the instrument parameters. The structure of each of these peptides was confirmed by mass spectrometry. The instrument parameters were optimized for each peptide (Supporting Information Table S1) and were also applied to the 15N-labeled peptide analogs which were used as internal

6



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

standards. The two analogs of the sheep peptide containing the amino acids at position 136 (M132LGSXMSRPL141; X = A or V) optimally fragmented in a similar fashion. The optimal fragments were the y8 (most intense), y6, and y5. Two homologous sheep peptides containing amino acids at position 154 (Y153XENMY158; X = H or R) optimally fragmented to yield the b5 (most intense) and b4 ions. Three sheep peptides containing the Y166RPVDXY172 (X = H, K, Q or R) fragmented to produce b5 (most intense for H, K, and Q), y3 (most intense for R), y5, and b4. Optimally fragmented peptide R159YPNQVY165 (peptide common to all of these PrP variants) yielded b6 (most intense) and b5 ions. The optimized instrument settings were used to quantify the amount of these peptides present in a sample. Distinguishing among the chymotryptic peptides. With such a large number of peptides it is possible that the signals from these peptides might overlap and confound the results. The precursor ions for most of the peptides were sufficiently different from one another to preclude interference. This was not the case for R159YPNQVY165 (m/z = 470.24), Y166RPVDKY172 (m/z = 470.73), and Y166RPVDQY172 (m/z = 470.73) where their precursor ions had a similar m/z and their product b6 ions had an identical m/z (758.39). R159YPNQVY165 was readily distinguished from Y166RPVDKY172 and Y166RPVDQY172 by using the b5 ion, since its m/z is 659.33 compared to an m/z of 631.31 for Y166RPVDKY172 and Y166RPVDQY172. Distinguishing between Y166RPVDKY172 and Y166RPVDQY172 relies on chromatographic differences since their parent ions and fragment ions are nearly identical. Thus all of the chymotryptic peptides could be readily distinguished from one another. The peptides M132LGSXMSRPL141 (X= A or V) and Y153XENMY158 (X = H or R) both contain the amino acid methionine. This amino acid can be converted to the sulfoxide by various enzymatic and artifactual oxidative processes.43, 54 The signal from an oxidized methionine would not be apparent using a method to detect only the unoxidized methionine. The MRM methods for these peptides were modified to include double or single methionine oxidation, respectively. The area ratios reported for these peptides include the sum of the unoxidized and oxidized forms of the peptide. By taking into account the oxidized methionines, this method provides an accurate estimate of the amount of each of these peptides. Preparing 15N-labeled internal standards. The required 15N-labeled sheep recombinant PrP was prepared by growing the clones expressing the sheep PrP polymorphisms in minimal medium supplemented with 15NH4Cl. This procedure yielded sheep PrP polymorphisms that were highly enriched with 15N. The 15N-labeled internal standards were derived from a chymotryptic digestion of each cloned sheep 15N-labeled PrP polymorphism. These peptides were analyzed by this MRM method to determine if any of the signals from a 15N-labeled peptide interfered with the signal of any other 15Nlabeled peptides. As predicted, none of the signals selected for this analysis from a 15Nlabeled peptide was observed to interfere with the analysis of other 15N-labeled peptides. The signal from the 15N peptides did not interfere with those of the unlabeled (14N) chymotryptic peptides, which indicated that the isotopic purity of the peptides was very high (> 99.7%). Quantifying peptide amounts in a sample using calibration curves. The amount of synthetic analyte peptide provided by the vendor was determined, where possible with peptides containing a tyrosine (T) and/or tryptophan (W), by measuring its

7


Page 8 of 20

Page 9 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


absorbance at 280 nm or by using the value provided by the vendor. The variation among these calculated values was less than 6%. The 15N-labeled internal standard peptides were obtained from the chymotryptic digest of the appropriate purified sheep PrP sample. For each of the nine synthetic peptides, a set of solutions was prepared. Each contained a fixed amount of the appropriate 15N-labeled internal standard and a set of dilutions (10, 20, 50, 500, 1000, or 10,000 attomole/injection) of the corresponding synthetic peptide. These solution sets were analyzed by mass spectrometry. The ratio of the area of a signal from an optimized ion of a synthetic peptide to that of a corresponding ion from its 15N-labeled analog were calculated (n=4) for each solution in the set (Figures S-1 to S-9, Supporting Information). Calibration curves were prepared for each of the peptides. These curves related the area ratio of the signal from the optimized ion from a known amount of synthetic peptide to the optimized ion from a fixed amount of the corresponding isotopically labeled internal standard. The curves were determined to be linear over a >100-fold range with excellent correlation coefficients (> 0.99). The peptide R159YPNQVY165 was present in all of the PrP samples. An additional set of calibration curves were prepared for each sheep peptide in a similar fashion, except that the area ratio of the 15N-labeled R159YPNQVY165 peptide was used in place of the 15 N-labeled internal standards. These data were used to generate calibration curves relative to the R159YPNQVY165 peptide (Figures S-10 to S17, Supporting Information). The relative calibration curves were linear over a > 100-fold range and the correlation coefficients were excellent (> 0.99). This means the amount of the peptides (136, 154, and 171) in a sample can be calculated relative to the R159YPNQVY165 peptide that is present in all digests and not just to an added 15N-labeled internal standard. This simplifies the analytical procedure, since only one internal standard is needed for all heterozygous samples. Limit of detection for sheep peptides. The nine synthetic peptides were serially diluted and independently analyzed using this MRM method. The signal intensity of the R159YPNQVY165 peptide is the most intense of all of the nine peptides (Figure S-18, Supporting Information). With the exception of the R159YPNQVY165 peptide, the other nine peptides have similar intensities. Signals are observed at injections of 20 to 100 attomoles. This indicates that the LOD is at least 50 attomoles (S/N > 3 x background) for these peptides. This also indicates that this method is very sensitive and will not require a large amount of sample. Quantitating the amount of PrPC in a Bolton PrPSc preparation. The method of Bolton et al. is a well established method of enriching a prion sample (> 95%) and contains both PK sensitive and PK resistant PrPSc.55 It does not, however, completely remove the PrPC from the sample. In order to determine the amount of residual PrPC present in the Bolton pellet, a sample of brain homogenate from a sheep (ARQ) uninfected with prions was subjected to a Bolton et al. preparation. The resulting pellet was digested with chymotrypsin and analyzed by this MRM method. The amount of PrPC in the Bolton pellet (50 mg brain tissue) was found to be 23 ± 5 fmol (n=2). The amount of PrPC reported to be present in 50 mg of sheep brain tissue is approximately 7 ± 1 picomole.21 This means that the Bolton procedure removes approximately 99.6% of the PrPC in the sample. The amount of PrPSc found in the Bolton pellet (50 mg spinal cord sample) for sample number 982, 983, 1696, or 1713 was 3.1 ± 0.1 pmol, 3.1 ± 0.3 pmol,

8



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1.2 ± 0.1 pmol, or 3.2 ± 0.1 pmol, respectively. The amount of residual PrPC expected to be present in the non PK treated PrPSc samples corresponds to between 0.7% and 2% of the total amount of PrPSc isolated from the scrapie-infected samples. This amount is also less than 0.5% of the total amount of PrPC present in the original brain tissue sample. We conclude that the amount of PrPC in the uninfected sample was detectable, but small when compared to the amount of PrPSc present in the four samples and would not to interfere with our analysis. This also means that it was not necessary to remove the PrPC with PK, which would also remove the PK-sensitive PrPSc present in the sample and, thereby, reduce the total amount of PrPSc available for analysis. Determining the extent of background interference with the chymotryptic peptides. Chymotryptic digestion of a complex sample will result in a large mixture of chymotryptic peptides. In order to interfere with this analysis, a molecule would have to have an identical chromatographic retention time and identical precursor and product ions. It was therefore necessary to determine if signals from any of these peptides interfered with signals from other peptides. The synthetic peptides were analyzed to determine if there was overlap with other peptides. The signals from pure chymotryptic peptides do not interfere with the analysis of the other chymotryptic peptides. It was necessary to determine whether any molecules present in the chymotryptic digestion of a pellet from an uninfected sheep might have signals that would interfere with this analysis. An ultracentrifuge pellet from an uninfected sheep brain (ARQ homozygote) was digested with chymotrypsin. The chymotryptic digestion of 15Nlabeled ARQ was added to the chymotryptic digest as the internal standard and the samples were analyzed by this MRM-based method (Figure 2). As noted previously, the pellet contained residual PrPC (ARQ/ARQ). Thus, the observed signals for the ARQ originated from the residual PrPC present in the sample. Examination of the chromatograms revealed that none of the signals other than those expected for the ARQ PrP was above noise. In conclusion, the chymotryptic digest does not produce molecules, other than those expected from the residual PrPC, which would significantly interfere with the analysis.

9


Page 10 of 20

Page 11 of 20

MRM Intensity

MRM Intensity

600

600

0

600

13

Alanine (A)

8

0 8

13

Valine (V)

13

154

0

Histidine (H)

MRM Intensity

600

600

0 13

1200

1200

MRM Intensity

1200

MRM Intensity

MRM Intensity

13

Arginine (R)

Retention time (min.)

136 1200

8

8

Histidine (H)


600

600

0

0 8

1200

MRM Intensity

1200

1200

1200

MRM Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

0 8

13

Lysine (K)

600

8

13

Glutamine (Q)

8

13

Arginine (R)


171 Figure 2. Chromatograms of molecules present in the chymotryptic digest of a Bolton pellet from an uninfected sheep brain homogenate (ARQ/ARQ genotype). Each is an MRM-based chromatogram, using the parameters for the indicated chymotrypic peptide polymorphism. Pink shaded columns indicate expected retention times of signals for the respective peptide polymorphism. The observed signals for the 136A (M132LGSAMSRPL141,), 154R (Y153RENMY158), and 171Q (Y166RPVDQY172) peptides are from the residual PrPC present in the sample. Optimizing the digestion period. PrPSc from scrapie-infected brain tissue (ARQ homozygote) was isolated by ultracentrifugation (Supporting Information). The resulting pellets were denatured and then digested with chymotrypsin for 1, 2, 4, or 20 hours at 30 ºC (Figure S-19, Supporting Information). These digests were analyzed by mass spectrometry to determine the optimal digestion period for PrPSc. The greatest peptide signal was detected after a 20 hour digestion. Overnight digestion was determined to be optimal for the three peptides M132LGSVMSRPL141, Y153RENMY158, and Y166RPVDQY172. This was also true for the R159YPNQVY165. Overnight digestion was selected as the optimal time for digestion. A mixture of 50 fmol of each of the synthetic peptides M132LGSAMSRPL141, M132LGSVMSRPL141, Y153HENMY158, Y153RENMY158, Y166RPVDHY172,

10



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Y166RPVDQY172, and Y166RPVDRY172 was digested with chymotrypsin overnight (n=3). This was done to determine the extent to which the peptides were degraded by the enzyme during the overnight digestion. Peptides M132LGSAMSRPL141, M132LGSVMSRPL141, Y166RPVDHY172, Y166RPVDQY172, and Y166RPVDRY172 showed no significant differences in the amount before and after overnight digestion (ttest; p > 0.05). The peptides Y153HENMY158 and Y153RENMY158 showed a significant difference between the starting amount and that present after overnight digestion (ttest; p > 0.05). This loss was similar (40%) for both the peptides, so it would not affect the calculations. Analysis of the data showed that the loss of the peptides due to digestion by chymotrypsin was less than the amount of the peptides produced by an overnight digestion. Origin of the sheep samples. All of the sheep samples came from flocks consisting of the Rasa Aragonesa breed56 that were naturally infected with scrapie (Table 1).57 The two sheep samples with the ARQ/VRQ genotype (982 and 983) originated from a flock of sheep (Flock 1; 151 genotyped) and the two samples with the ARQ/ARH (1696 and 1713) genotype came from a different flock of 785 animals (Flock 2; all genotyped). Based on the incomplete genotyping of flock 1, ARQ/ARQ was the predominant genotype (~61%) and A at position 136 was the predominant (84%) polymorphism at that position. Approximately 62% of the scrapie cases from flock 1 possessed the ARQ/ARQ genotype. ARQ/VRQ heterozygotes accounted for less than 10% of flock 1, but 1/3 of the scrapie cases. Approximately 24% of this flock was homozygous or heterozygous for the protective ARR allele. In the second flock, ARQ homozygotes comprised approximately 39% of the sheep and 63% of the scrapie cases. Animals possessing at least one protective ARR allele accounted for 1/3 of the total. ARQ/ARH heterozygotes accounted for approximately 13% of flock 2 and 29% of the scrapie cases. Approximately 87% of the sheep in flock 2 that were not heterozygous for H and Q at position 171 were homozygous for Q or heterozygous for Q and R at position 171.

11


Page 12 of 20

Page 13 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Genotypes of the flocks from which the scrapie infected spinal cord tissue originated.

Flock 1§

Flock 2ǂ

Number of Genotype uninfected sheep by genotype

Number of Scrapieinfected sheep by genotype

ARQ/ARH 8 1 ARQ/ARQ 92 39 ARQ/VRQ 14 21 ARR/ARH 3 --ARR/ARQ 21 --ARR/ARR 3 --ARR/VRQ 9 --VRQ/VRQ 1 1 ARH/ARH --1 Total 151 63 § The genotyping of uninfected sheep in flock 1 is incomplete. The genotyping of the scrapie-infected sheep from flock 1 is complete. ǂ The genotyping of both scrapie-infected and uninfected sheep from flock 2 is complete.

Number of Number Genotype uninfected of sheep by Scrapiegenotype infected sheep by genotype AHQ/ARH AHQ/VRQ ARH/ARH ARH/VRQ ARK/ARQ ARK/ARR ARQ/AHQ ARQ/ARH ARQ/ARQ ARQ/VRQ ARR/AHQ ARR/ARH ARR/ARQ ARR/ARR ARR/VRQ VRQ/VRQ Total

3 6 14 12 3 2 9 103 304 67 3 42 161 27 26 3 785

----6 --------36 79 3 ----------1 125

Quantifying the relative contribution of the PrP polymorphisms to the PrPSc isolated from a heterozygous animal. Four CNS samples from the spinal cord near the obex of heterozygous sheep were analyzed. Each of the animals was from a flock of naturally scrapie infected sheep. The spinal cord samples were homogenized and subjected to ultracentrifugation in non denaturing detergent according to the method of Bolton et al. where pellets resulting from the procedure contain both PK sensitive and PK resistant PrPSc. One set (n=4) of four pellets was denatured and then analyzed by this MRM method to determine the amount of total PrPSc. Another set of four pellets was treated with proteinase K to remove the PK sensitive PrPSc and then denatured before being analyzed this MRM method (n=2). The result of this analysis is shown in Table 2.

12



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 2. Percentage of PrP polymorphisms present in combined PK-sensitive and PKresistant PrPSc (-PK), or PK-resistant PrPSc (+PK). Sample▲

+ or PK

1696 -PK +PK 1713 -PK +PK 982 -PK +PK 983 -PK +PK

Polymorphism 171H 63 ± 4 63 ± 5 171H 58 ± 5 54 ± 1

171Q 37 ± 4 37 ± 5 171Q 42 ± 5 46 ± 1

136A 69 ± 6 72 ± 1 136A 70 ± 6 73 ± 2

136V 31 ± 6 28 ± 1 136V 30 ± 6 27 ± 2

▲ Samples 1696 and 1713 are from different heterozygous sheep (ARH/ARQ) from flock 2. Samples 982 and 983 are from different heterozygous sheep (ARQ/VRQ) from flock 1. In all four of these heterozygous samples both polymorphisms are found in either the PK-sensitive plus PK-resistant fraction or the PK-resistant fraction. With the exception of sample 1713, all of the samples showed no statistical difference between the PK treated and non-PK treated PrPSc (ttest; p > 0.05). In the 982 and 983 samples the A136 predominates, even though the V136 is considered to be the more susceptible polymorphism at position 136. The A136 polymorphism predominates in both the sheep in flock 1 and those infected with scrapie, suggesting that the prions causing scrapie in this flock carried the A136 polymorphism. The 1696 and 1713 samples both contain PrPSc with Q171 and H171 albeit in different ratios. The proportion of H171 is greater than Q171, even though Q171 predominates in flock 2 and in the scrapie cases from flock 2. It is also possible that the prion or prions causing scrapie in these flocks were polymorphic. These results, in any case, indicate that the PrPSc from heterozygotes is composed of both polymorphisms, which suggests a possible role of heterozygous animals in expanding the host range of prions. Conclusions We have developed a method of quantifying the relative amount of the PrP polymorphisms present in PrPSc from a heterozygous sheep using a single proteolytic enzyme, chymotrypsin. This approach can be used on both the PK-sensitive and PKresistant forms of PrPSc. It is sufficiently sensitive as to require as little as 5mg of brain 13


Page 14 of 20

Page 15 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


or CNS tissue from a prion-infected sheep. Using this method we determined that PrPSc from PrP heterozygous sheep naturally infected with prions contained both polymorphisms. Furthermore, the relationship between the optimized signal intensity of peptides containing positions 136, 154, and 171 are linear with the optimized signal from the peptide R159YPNQVY165, so this method does not require the use of an isotopically labeled internal standard. This method can be applied to the PrPSc present in heterozygous animals that have been naturally or experimentally infected with prions. Surprisingly, the polymorphism more resistant to scrapie (A at 136 or H at 171) was the majority in all four of the samples. Furthermore, it was the majority for samples containing both PK-sensitive and PK-resistant PrPSc and only PK-resistant PrPSc. In other studies of heterozygous animals, the more resistant polymorphism was found in lower abundance.22, 24, 25 This indicates that heterozygous animals have the capacity to convert both PrPC polymorphisms into PrPSc, albeit in different proportions. In the future we want to analyze samples from heterozygous animals that have been experimentally infected with the same prion. Digestion of the sheep PrP (ARQ) with chymotrypsin yielded the peptide G94QGGSHSQW102 a peptide analogous to one produced by the chymotryptic digestion of deer PrP (G94QGGTHSQW102). Polymorphisms at position 95 (H) and 96 (G or S) are important in determining the course of chronic wasting disease, a prion disease of cervids.58 This suggests that this approach is broadly applicable to the study of other prion-infected species, including heterozygous deer infected with chronic wasting disease. Supporting Information Table of optimized instrument parameters, text with supporting experimental procedures, figures of calibration curves, figure for limit of detection, and figure showing relative amount of peptides after digestion with chymotrypsin (PDF) Notes The authors declare no competing financial interest.

Acknowledgments This work was supported by the United States Department of Agriculture (CRIS 203032000-009-00 and 5030-32000-103-00) and the government of Spain (Ministerio de Economia y Competitividad grants AGL2015-65560-R and BFU2013-48436-C2-1-P). Mention of a commercial product does not reflect a recommendation or endorsement of that product by the USDA. The USDA is an equal opportunity provider and employer.

References.

14



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(1) (2) (3)

(4) (5)

(6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22)

Hunter, N.; Goldman, W.; Marshall, E.; O'Niell, G. Arch. Virol. Suppl. 2000, 16, 181-188. Prusiner, S. B. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 13363-13383. Pattison, I. H. In Slow, Latent and Temperate Virus Infections (NINDB Monograph 2); Gajdusek, C. J., Gibbs, C. J., Alpers, M. P., Eds.; U.S. Government Printing Office: Washington D. C., 1965, pp 249-257. Belt, P. B.; Muileman, I. H.; Schreuder, B. E.; Bos-de Ruijter, J.; Gielkens, A. L.; Smits, M. A. J. Gen. Virol. 1995, 76 ( Pt 3), 509-517. Clouscard, C.; Beaudry, P.; Elsen, J. M.; Milan, D.; Dussaucy, M.; Bounneau, C.; Schelcher, F.; Chatelain, J.; Launay, J. M.; Laplanche, J. L. J. Gen. Virol. 1995, 76 ( Pt 8), 2097-2101. Hunter, N.; Goldmann, W.; Smith, G.; Hope, J. Arch. Virol. 1994, 137, 171-177. Westaway, D.; Zuliani, V.; Cooper, C. M.; Da Costa, M.; Neuman, S.; Jenny, A. L.; Detwiler, L.; Prusiner, S. B. Genes Dev. 1994, 8, 959-969. Hunter, N.; Foster, J. D.; Goldmann, W.; Stear, M. J.; Hope, J.; Bostock, C. Arch. Virol. 1996, 141, 809-824. Ikeda, T.; Horiuchi, M.; Ishiguro, N.; Muramatsu, Y.; Kai-Uwe, G. D.; Shinagawa, M. J. Gen. Virol. 1995, 76 ( Pt 10), 2577-2581. Goldmann, W.; Hunter, N.; Smith, G.; Foster, J.; Hope, J. J. Gen. Virol. 1994, 75 ( Pt 5), 989-995. Goldmann, W.; Hunter, N.; Benson, G.; Foster, J. D.; Hope, J. J. Gen. Virol. 1991, 72 ( Pt 10), 2411-2417. Hunter, N.; Goldmann, W.; Benson, G.; Foster, J. D.; Hope, J. J. Gen. Virol. 1993, 74 ( Pt 6), 1025-1031. Baylis, M.; Goldmann, W.; Houston, F.; Cairns, D.; Chong, A.; Ross, A.; Smith, A.; Hunter, N.; McLean, A. R. J. Gen. Virol. 2002, 83, 2907-2914. Baylis, M.; Houston, F.; Goldmann, W.; Hunter, N.; McLean, A. R. Proc. Biol. Sci. 2000, 267, 2029-2035. Luhken, G.; Buschmann, A.; Brandt, H.; Eiden, M.; Groschup, M. H.; Erhardt, G. Vet. Res. 2007, 38, 65-80. Arsac, J. N.; Andreoletti, O.; Bilheude, J. M.; Lacroux, C.; Benestad, S. L.; Baron, T. Emerg. Infect. Dis. 2007, 13, 58-65. Moum, T.; Olsaker, I.; Hopp, P.; Moldal, T.; Valheim, M.; Moum, T.; Benestad, S. L. J. Gen. Virol. 2005, 86, 231-235. Saunders, G. C.; Cawthraw, S.; Mountjoy, S. J.; Hope, J.; Windl, O. J. Gen. Virol. 2006, 87, 3141-3149. Greenlee, J. J.; Zhang, X.; Nicholson, E. M.; Kunkle, R. A.; Hamir, A. N. J. Vet. Diagn. Invest. 2012, 24, 554-558. Kitamoto, T.; Iizuka, R.; Tateishi, J. Biochem. Biophys. Res. Commun. 1993, 192, 525-531. Moudjou, M.; Frobert, Y.; Grassi, J.; La Bonnardiere, C. J Gen Virol 2001, 82, 2017-2024. Morel, N.; Andreoletti, O.; Grassi, J.; Clement, G. Rapid Commun. Mass Spectrom. 2007, 21, 4093-4100.

15


Page 16 of 20

Page 17 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23)

(24) (25)

(26) (27)

(28) (29)

(30) (31) (32) (33) (34) (35) (36)

(37) (38) (39) (40) (41)

(42)

Bilheude, J. M.; Brun, A.; Morel, N.; Diaz San Segundo, F.; Lecroix, S.; Espinosa, J. C.; Gonzalez, L.; Steele, P.; Grassi, J.; Andreoletti, O.; Torres, J. M. J. Virol. Methods 2007, 145, 169-172. Langeveld, J. P.; Jacobs, J. G.; Hunter, N.; van Keulen, L. J.; Lantier, F.; van Zijderveld, F. G.; Bossers, A. J. Virol. 2016, 90, 805-812. Jacobs, J. G.; Bossers, A.; Rezaei, H.; van Keulen, L. J.; McCutcheon, S.; Sklaviadis, T.; Lantier, I.; Berthon, P.; Lantier, F.; van Zijderveld, F. G.; Langeveld, J. P. J. Virol. 2011, 85, 12537-12546. Moore, R. A.; Head, M. W.; Ironside, J. W.; Ritchie, D. L.; Zanusso, G.; Choi, Y. P.; Priola, S. A. PLoS Pathog. 2016, 12, e1005416. Stahl, N.; Baldwin, M.; Teplow, D. B.; Hood, L. E.; Beavis, R.; Chait, B.; Gibson, B. W.; Burlingame, A. L.; Prusiner, S. B. In Prion diseases of humans and animals; Prusiner, S. B., Collinge, J., Powell, J., Anderton, B., Eds.; Ellis Horwood: New York, 1992, pp 361-379. Stahl, N.; Baldwin, M. A.; Teplow, D. B.; Hood, L.; Gibson, B. W.; Burlingame, A. L.; Prusiner, S. B. Biochemistry 1993, 32, 1991-2002. Rudd, P. M.; Endo, T.; Colominas, C.; Groth, D.; Wheeler, S. F.; Harvey, D. J.; Wormald, M. R.; Serban, H.; Prusiner, S. B.; Kobata, A.; Dwek, R. A. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 13044-13049. Endo, T.; Groth, D.; Prusiner, S. B.; Kobata, A. Biochemistry 1989, 28, 83808388. Stimson, E.; Hope, J.; Chong, A.; Burlingame, A. L. Biochemistry 1999, 38, 4885-4895. Stahl, N.; Borchelt, D. R.; Hsiao, K.; Prusiner, S. B. Cell 1987, 51, 229-240. Stahl, N.; Baldwin, M. A.; Prusiner, S. B. Cell Biol. Int. Rep. 1991, 15, 853-862. Stahl, N.; Baldwin, M. A.; Hecker, R.; Pan, K. M.; Burlingame, A. L.; Prusiner, S. B. Biochemistry 1992, 31, 5043-5053. Prusiner, S. B.; McKinley, M. P.; Bowman, K. A.; Bolton, D. C.; Bendheim, P. E.; Groth, D. F.; Glenner, G. G. Cell 1983, 35, 349-358. Sajnani, G.; Silva, C. J.; Ramos, A.; Pastrana, M. A.; Onisko, B. C.; Erickson, M. L.; Antaki, E. M.; Dynin, I.; Vazquez-Fernandez, E.; Sigurdson, C. J.; Carter, J. M.; Requena, J. R. PLoS Pathog. 2012, 8, e1002547. Safar, J.; Wille, H.; Itri, V.; Groth, D.; Serban, H.; Torchia, M.; Cohen, F. E.; Prusiner, S. B. Nat. Med. 1998, 4, 1157-1165. Pastrana, M. A.; Sajnani, G.; Onisko, B.; Castilla, J.; Morales, R.; Soto, C.; Requena, J. R. Biochemistry 2006, 45, 15710-15717. Silva, C. J. Prion 2014, 8, 42-50. Vazquez-Fernandez, E.; Alonso, J.; Pastrana, M. A.; Ramos, A.; Stitz, L.; Vidal, E.; Dynin, I.; Petsch, B.; Silva, C. J.; Requena, J. R. PLoS One 2012, 7, e50111. Chesebro, B.; Trifilo, M.; Race, R.; Meade-White, K.; Teng, C.; LaCasse, R.; Raymond, L.; Favara, C.; Baron, G.; Priola, S.; Caughey, B.; Masliah, E.; Oldstone, M. Science 2005, 308, 1435-1439. Chesebro, B.; Race, B.; Meade-White, K.; Lacasse, R.; Race, R.; Klingeborn, M.; Striebel, J.; Dorward, D.; McGovern, G.; Jeffrey, M. PLoS Pathog. 2010, 6, e1000800.

16



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(43) (44)

(45) (46) (47) (48) (49) (50) (51) (52)

(53) (54) (55) (56) (57)

(58)

Silva, C. J.; Onisko, B. C.; Dynin, I.; Erickson, M. L.; Vensel, W. H.; Requena, J. R.; Antaki, E. M.; Carter, J. M. Biochemistry 2011, 49, 1854-1861. Onisko, B. C.; Silva, C. J.; Dynin, I.; Erickson, M.; Vensel, W. H.; Hnasko, R.; Requena, J. R.; Carter, J. M. Rapid Commun. Mass Spectrom. 2007, 21, 40234026. Onisko, B.; Dynin, I.; Requena, J. R.; Silva, C. J.; Erickson, M.; Carter, J. M. J. Am. Soc. Mass Spectrom. 2007, 18, 1070-1079. Silva, C. J.; Onisko, B. C.; Dynin, I.; Erickson, M. L.; Requena, J. R.; Carter, J. M. Anal. Chem. 2011, 83, 1609-1615. Sturm, R.; Sheynkman, G.; Booth, C.; Smith, L. M.; Pedersen, J. A.; Li, L. J. Am. Soc. Mass Spectrom. 2012, 23, 1522-1533. Gielbert, A.; Davis, L. A.; Sayers, A. R.; Hope, J.; Gill, A. C.; Sauer, M. J. J. Mass. Spectrom. 2009, 44, 384-396. Silva, C. J.; Erickson-Beltran, M. L.; Dynin, I. C. Biochemistry 2016, 55, 894902. Sun, G.; Anderson, V. E. Electrophoresis 2004, 25, 959-965. Swiderek, K. M.; Davis, M. T.; Lee, T. D. Electrophoresis 1998, 19, 989-997. Munoz-Montesino, C.; Sizun, C.; Moudjou, M.; Herzog, L.; Reine, F.; Chapuis, J.; Ciric, D.; Igel-Egalon, A.; Laude, H.; Beringue, V.; Rezaei, H.; Dron, M. J. Virol. 2016, 90, 6963-6975. Dumpitak, C., Heinrich-Heine-Universität, Düsseldorf, 2003. Silva, C. J.; Dynin, I.; Erickson, M. L.; Requena, J. R.; Balachandran, A.; Hui, C.; Onisko, B. C.; Carter, J. M. Biochemistry 2013, 52, 2139-2147. Bolton, D. C.; Rudelli, R. D.; Currie, J. R.; Bendheim, P. E. J. Gen. Virol. 1991, 72, 2905-2913. Mason, I. L. In Genetics resources of pig, sheep and goat; Maijala, K., Ed.; Elsevier: Amsterdam, Netherlands, 1991, pp 556. Acin, C.; Martin-Burriel, I.; Goldmann, W.; Lyahyai, J.; Monzon, M.; Bolea, R.; Smith, A.; Rodellar, C.; Badiola, J. J.; Zaragoza, P. J. Gen. Virol. 2004, 85, 21032110. Duque Velasquez, C.; Kim, C.; Herbst, A.; Daude, N.; Garza, M. C.; Wille, H.; Aiken, J.; McKenzie, D. J. Virol. 2015, 89, 12362-12373.

17


Page 18 of 20

Page 19 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


H G W Q P G G G P H G G G G G G G G S P G R P Q G G G H G W Q S H G W Q P G G W Q G N Y P G G W Q P G G G G H L G V G A A A H M T P S K W Q P R G T W G P P R S V K N K K P N Q G A A V A G K Y K G S G N G G G Y L S M R L H G K 25 N H2 G M G A S P I 232 A F N V O P G D V Y 136 R N Y D 171 Q R Q Y Q Y N H P R Y O E Y S Y PO A R D Y Q N Y Y R S OH Q E M N R Q E N 154 N F Y V HO P O O P OH H T Q D O O I C sC V H2N M s N Q I OO E V T O V V K R Q Octarepeat GPI E M H T -Sheet V anchor K I T I -Helix D T T T Sugar residue E T T G K F N E Sugar

antennae



MRM Intensity

MRM Intensity

600

600

0

600

13

8

Alanine (A)

0 8

13

Valine (V)

13

8


136

154

0

600

0 8

13

Histidine (H)

MRM Intensity

600

1200

1200

MRM Intensity

MRM Intensity

1200

600

13

Lysine (K)

600

0

0 8

13

Arginine (R)

Histidine (H)


1200

MRM Intensity

600

0

0 8

1200

MRM Intensity

1200

1200

1200

MRM Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 20 of 20

8

13

Glutamine (Q)


171 ACS Paragon Plus Environment

8

13

Arginine (R)

Quantitating PrP Polymorphisms Present in Prions from Heterozygous

Recommend Documents