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Food Forensics: Using Mass Spectrometry to Detect Foodborne Protein Contaminants, as Exemplified by Shiga Toxin Variants and Prion Strains. Christopher J. Silva J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01517 • Publication Date (Web): 02 Jun 2018 Downloaded from http://pubs.acs.org on June 2, 2018

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Journal of Agricultural and Food Chemistry

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Food Forensics: Using Mass Spectrometry to Detect Foodborne Protein Contaminants, as

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Exemplified by Shiga Toxin Variants and Prion Strains.

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Christopher J. Silva*

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Produce Safety & Microbiology Research Unit, Western Regional Research Center, United

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States Department of Agriculture, Agricultural Research Service, Albany, California 94710,

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United States of America.

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* Corresponding Author (Tel: 510.559.6135; Fax: 510.559.6429; E-mail:

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[email protected])

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ACS Paragon Plus Environment


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Abstract

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Food forensicists need a variety of tools to detect the many possible food contaminants. Due to

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its analytical flexibility, mass spectrometry is one of those tools. Use of the multiple reaction

28

monitoring (MRM) method expands its use to quantitation as well as detection of infectious

29

proteins (prions) and protein toxins such as Shiga toxins.

30

inactivate prions and Shiga toxins; the proteins are digested with proteases to yield peptides

31

suitable for MRM-based analysis. Prions are detected by their distinct physicochemical

32

properties and by differential covalent modification. Shiga toxin analysis is based on detecting

33

peptides derived from the five identical binding B subunits comprising the toxin.

34

internal standards are prepared from cloned proteins. These examples illustrate the power of

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MRM, in that the same instrument can be used to safely detect and quantitate protein toxins,

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prions and small molecules that might contaminate our food.

37 38 39 40 41 42 43 44 45 46


The sample processing steps

15

N-labeled

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Introduction

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As food comes from more diverse sources, in an ever-astonishing variety, and is

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processed to widely varying extents, the possibility for deliberate or inadvertent food

51

manipulation or contamination also increases. As a result, food forensicists are faced with

52

substantial and evolving challenges. A common form of manipulation is economic substitution

53

of more expensive food with less expensive, but similar food. Seafood harvested from around

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the world and often sold to consumers in locations far removed from its harvest is sometimes

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mislabeled accidentally or deliberately.1

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species, such as salmon, is both farmed and wild-harvested. In Europe, marginally cheaper horse

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meat has been mislabeled as modestly more expensive beef or pork.2 Olive oil is sometimes

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adulterated with other cheaper vegetable oils.3-4 It is not surprising that food forensicists would

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need an equally diverse set of tools to test for food substitution.5

Accurate labeling is further complicated when a

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Adulteration for unintentional, natural or economic reasons can cause more serious

61

problems. In China, melamine (Figure 1) was used to confound the Kjeldahl-based protein

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analysis to make milk appear more concentrated than it actually was.6-8 Turmeric produced in

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India or Bangladesh and sold in the United States was discovered to be adulterated with lead (II)

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chromate to enhance the color of and increase the weight of the product.9 Ciguatoxin 1B (Figure

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1) is an example of a large and structurally complicated marine toxin that may naturally

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contaminate a variety of seafoods.10 In addition to specific chemicals, food may be contaminated

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with other small molecule toxins, infectious proteins (prions), protein toxins or bacteria that

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produce toxins.11-13 The variety of possible contaminants means that no universal analytical

69

method can be used by a forensicist to detect all possible contaminants.5



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The cost of food adulteration or contamination is substantial, both in terms of human

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suffering and economic losses. The consequences of melamine adulteration to the Chinese dairy

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industry and the Chinese economy in general have been substantial.14 The human consequences

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are greater; approximately 294,000 children were sickened, 50,000 required hospitalization and

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at least 6 died.14 In the United States approximately 175,000 cases of Shiga toxin-producing E.

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coli (STEC) occurred yearly from 2000-2008.15 The economic cost of a single outbreak in 2006

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was estimated to be $200 million (2006 $US).16 The STEC outbreak that occurred in 1992 cost

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the company $160 million in lost sales (1992 $US) and a 30% drop in its stock price.17 More

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than 8300 school children were affected by the 1996 STEC outbreak in Sakai, Japan.18 In 2011,

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an STEC outbreak in Germany sickened more than 3800 people.19

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encephalopathy (BSE), a prion disease of domestic cattle, devastated British agriculture.20 Its

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human form, variant Creutzfeldt-Jakob disease (vCJD), has killed at least 230 people.21 It is

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clear that in addition to being challenging, food forensics is crucial to human health.

83

Bovine spongiform

In practice, mass spectrometry is a multiplex analytical tool that is used to detect an

84

astonishingly diverse range of contaminants.11

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contaminants, such as melamine and a suite of pesticide residues.22

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aflatoxin B1, microcystin-LR, tetrodotoxin, ciguatoxin 1B, and other shellfish toxins (Figure 1)

87

are also readily detectable by mass spectrometry.13, 23-26 The development of newer ionization

88

methods has permitted the detection of whole proteins by mass spectrometry. Sector instruments

89

allow for the “filtering” of molecules based on their molecular weights. The multiple reaction

90

monitoring (MRM) method exploits these properties to allow the detection and quantitation of

91

small amounts of specific small molecules in complex mixtures.27 The MRM method is not

92

suited for the direct analysis of proteins, but it can be used to analyze peptides derived from

It has been used to detect small molecule


Mycotoxins, such as

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those proteins after they have been digested with proteases, such as trypsin or chymotrypsin.

94

This also provides greater confidence that a protein has been correctly identified when more than

95

one peptide derived from it can be detected. Mass spectrometry can potentially provide more

96

information about potential food contaminants than any other method.

97

Prions represent a novel detection challenge, since they are infectious proteins.28 The

98

research standard for prion detection is the bioassay.29 It can be used to establish that the sample

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material is infectious and that it causes the lesions characteristic of a prion disease.30 Incubation

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times for prion diseases vary from two months to several years, which precludes using bioassay

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as a routine detection method.29 Cell-based systems have been used to amplify prions, but not all

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prions amplify in cell culture.31

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hematoxylin and eosin stained fixed tissue slice, or the presence of prions stained by

104

immunohistochemistry, are also diagnostics used by regulatory agencies to demonstrate the

105

presence of prions and the damage that they cause in sheep and domestic cattle.

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Unfortunately, pathological changes are only observable in the later stages of the disease.34-35

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Western blotting is also used by regulatory agencies to rapidly diagnose prion diseases.32-33

108

Prions have been detected using prion amplification methods related to protein misfolding cyclic

109

amplification (PMCA).36 Not all prions amplify using these approaches and prion amplification

110

techniques also spontaneously produced infectious prions from bacterially derived recombinant

111

PrP (rPrP).37 Mass spectrometry remains the most sensitive means of directly detecting prions,

112

without the use of bioassay.38 In principle, mass spectrometry can also be used to detect the

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prions amplified by techniques such as PMCA sooner, thereby permitting more rapid detection

114

of prions.

The presence of appropriate vacuolization (spongiform) in a


32-33


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Shiga toxins are large multi-component toxins produced by phage-infected bacterial food

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contaminants. There are a variety of means to detect Shiga toxins to determine the source of

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contamination.39 Regulatory agencies focus on the E. coli serotypes (vide infra) associated with

118

toxin production.40-41 Shiga toxins have been detected using animal bioassay and cell assay.42-45

119

While these methods are sensitive, they cannot distinguish among the possible Shiga toxins,

120

unless the molar amount of Shiga toxin present in the sample is already known. Shiga toxin

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production is also inferred by the presence of specific virulence genes.46

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methods have been widely used to detect and distinguish among the Shiga toxins.47-56 To detect

123

any new Shiga toxins using antibodies, new antibodies need to be developed. Top down mass

124

spectrometry is also used to detect Shiga toxins. 57-62 An MRM-based approach has advantages

125

over these other methods in that it can be used to both distinguish among types and variants of

126

Shiga toxins and quantitate them (vide infra).63-64

Antibody based

127

MRM can also be used to analyze proteins following digestion with proteases, such as

128

trypsin or chymotrypsin, to yield a characteristic set of small molecule peptides.27 By using this

129

approach, forensicists can apply mass spectrometry to protein toxins such as Shiga toxins, which

130

are enzymatic toxins.

131

pathogenic proteins whose pathology is enciphered in their conformation. Identifying Shiga

132

toxins represents a challenge to detect and distinguish among closely related known proteins and

133

to adapt that technique to the detection of novel Shiga toxins as they are discovered. In the case

134

of prions, the challenge is to detect and distinguish among protein conformations. How MRM

135

has been used to accomplish both goals is described.

In addition, they can use MRM to detect the presence of prions,

136 137

Multiple reaction monitoring (MRM) method


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The multiple reaction monitoring method is a powerful means of analyzing the peptides

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derived from proteins.27 MRM requires the use of sector instruments, which are expensive and

141

not well suited for high throughput sample processing.

142

identification of suitable peptides, which necessitates an initial qualitative proteomic analysis to

143

identify peptides suitable for MRM-based analysis. In addition, synthetic peptides need to be

144

prepared and stable-isotope internal standards acquired (vide infra).

145

methods have the distinct advantage of greater sensitivity and flexibility. The MRM method

146

directly detects molecules, provides more information than is available by other means, and can

147

be used to simultaneously detect a variety of diverse molecules (multiplex).

148

power of triple quadrupole instruments to apply mass filters which permit selected ions to pass

149

on to subsequent sectors and thereby detect only ions that have the user defined properties, while

150

discarding the others (Figure 2). Modern sector instruments have cycling times (milliseconds)

151

that are much faster than chromatographic elution times (seconds). This means that the rapid

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sequential analysis of multiple ions appears to be simultaneous as samples elute from the

153

column. In this way a stable isotope-labeled internal standard can be used to demonstrate that an

154

unknown molecule shares the same physicochemical properties, e.g., chromatographic retention

155

time and characteristic fragmentation, as the known stable isotope-labeled molecule. A cartoon

156

of the triple quadrupole mass spectrometer and the multiple reaction monitoring method is in

157

Figure 3. The one drawback to this method is that it detects small molecules and not proteins.

The MRM method requires the

However, MRM-based

It exploits the

158

Proteins, however, can be analyzed by MRM, provided that they are cleaved into shorter

159

peptides prior to analysis. This is usually accomplished by digestion with a protease, such as

160

trypsin. Such procedures are now automated. They involve the reduction of any disulfide bonds



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with a reducing agent, such as dithiothreitol (DTT). The now free thiol groups (S-H) are reacted

162

with an alkylating agent, such as iodoacetamide, to yield the corresponding carbamidomethyl

163

group (S-CH2-CO-NH2). The covalent carbamidomethyl modification prevents the reformation

164

of disulfide bonds. The reduced and alkylated protein is then digested with a protease such as

165

trypsin to yield a set of tryptic peptides.

166

phosphines, have been used instead of DTT.66-67 Alkylating agents other than iodoacetamide can

167

be used covalently modify free thiols.68

168

variety of other proteases, such as chymotrypsin or thermolysin.69 Proteins can also be cleaved

169

by reagents such as cyanogen bromide. This variety of tools can be exploited to analyze specific

170

proteins.70

Other reducing agents, such as water soluble

Reduced and alkylated proteins can be cleaved by a

171

The resulting set of peptides is analyzed by a qualitative proteomic analysis to identify

172

peptides suitable for MRM analysis. Such peptides usually have molecular weights between 500

173

and 2,000. Those possessing the most intense signals are candidates for an MRM based analysis.

174

Physicochemical properties of the peptides need to be considered. For example, methionines can

175

be oxidized to their corresponding sulfoxides and N-terminal glutamines (and to a lesser extent

176

glutamate) can spontaneously form N-terminal pyroglutamates in solution.

177

transformations would result in the signal of a single peptide to be spread among two or more

178

different chemical analogs of that peptide.

Such chemical

179

Once a set of suitable peptides has been identified, the instrument parameters for the

180

triple quadrupole can be optimized for each peptide. The precursor ion is determined from the

181

qualitative analysis.

182

increasing the collision energy of the carrier gas and monitoring the resulting fragmentation of

183

the peptide. The values m/z of the most intense product ions become the mass filters for the third

The optimal fragmentation conditions are determined empirically by


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quadrupole.

This process is repeated for the remaining peptides.

This results in the

185

identification of an analyte peptide, which is the one with the most intense signal. The other

186

peptides can be used to confirm the presence of the progenitor protein. The protease digestion

187

yields a characteristic set of peptides, which can be used to quantitate and confirm the presence

188

of the progenitor protein, often in the attomole (10-18 mole) range.

189

Standard molecular biology techniques can be used to conveniently generate stable

190

isotope-labeled internal standards for the peptides optimized for MRM analysis.63, 71 A desired

191

gene is cloned into an appropriate vector and overexpressed in a suitable E. coli host. This

192

permits the convenient production of proteins and permits the generation of internal standards. If

193

the E. coli host is grown in minimal medium supplemented with an essential nutrient that is

194

highly enriched (99.7%) with 15N (e.g., such as 15NH4Cl), then all the nitrogen-bearing molecules

195

synthesized by that E. coli will also be highly enriched with

196

(14N) nitrogen. Digestion of purified proteins produced in this way would yield

197

peptides enriched with the 15N-label. The extent of isotopic incorporation is dependent upon the

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isotopic purity of the 15NH4Cl and the number of nitrogens present in the peptide. Peptides used

199

in the work cited in this review contain between 10 and 21 nitrogens. Between 96 and 93%

200

(respectively) of such peptides would be expected to contain exclusively 15N, with the remainder

201

comprising molecules containing mostly 15N, save for one 14N. Thus, 15N-enriched peptides can

202

be used as internal standards to identify and quantitate the peptides present in a sample.

15

N instead of natural abundance 15

N-labeled

203

The mass filter settings will exclude the portion of molecules containing the various

204

natural abundance stable isotopes. The proportion of internal standard peptides or peptide

205

fragments containing one 14N will be excluded by these mass filters. Analyte peptides derived

206

from samples containing naturally occurring

13

C (~1% natural abundance) are similarly



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excluded. The calibration curves establish an empirical relationship between synthetic analyte

208

peptides containing only

209

digestion of the isotopically (15N) enriched proteins, containing only

210

relationships are linear over a 100-fold range and have excellent correlation coefficients.

12

C and the internal standard peptides, derived from the protease 15

N. In practice, such

211 212

Prions

213 214

Prions are pathological proteins that cause transmissible spongiform encephalopathy

215

(TSE), a fatal neurological disease.28,

72

216

incubation, and a comparatively short symptomatic period, followed by death. The human TSEs

217

are Creutzfeldt-Jakob disease (CJD),73-74 kuru,75 Gerstmann–Sträussler–Scheinker (GSS)

218

disease,76 and fatal familial insomnia (FFI).77-78

219

spongiform encephalopathy (BSE) when they consume BSE-contaminated feed.79 BSE is the

220

only known zoonotic prion disease. The human form of BSE is called variant Creutzfeldt-Jakob

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(vCJD) disease.80 Sheep scrapie is transmitted among domestic sheep and is the oldest known

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TSE, first described in the middle of the 18th century.81-82 Transmissible mink encephalopathy

223

(TME) is a prion disease transmitted to farmed mink by contaminated feed; the last outbreak of

224

TME was in 1985.83 Chronic wasting disease (CWD) is a prion disease of cervids (white-tailed

225

deer, mule deer, elk, moose, European moose, red deer, and reindeer) and the only one naturally

226

transmitted among wild animals.84-86 More recently a camel prion disease (CPD) has been

227

discovered in Algeria that appears to be transmitted among domesticated camels in a manner

228

analogous to that of CWD and not through feed.87

TSEs are characterized by a long asymptomatic

Domestic cattle are infected with bovine


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Prions (PrPSc) cause disease by inducing a normal cellular prion protein (PrPC) to adopt

230

the prion conformation.71,

89-90

Both PrPC and PrPSc possess identical covalent structures and

231

only differ in their conformation.89 PrPC is a highly conserved monomeric protein of uncertain

232

function (Figure 4).91-92 It is soluble in non-denaturing detergents and its secondary structure is

233

comprised of α-helices, unstructured regions, and a small amount of β-sheet (Figure 5).93 In

234

contrast, the pathogenic (PrPSc) conformation is multimeric, insoluble in non-denaturing

235

detergents, and has no known biological function (Figure 5).28 A large body of spectroscopic

236

evidence supports the hypothesis that the secondary structure of PrPSc is composed of β-sheet

237

and unstructured regions, with no α-helices.94 Additional spectroscopic evidence suggests that

238

the β-sheet secondary structure is contained in a tertiary β-helical structure known as a four rung

239

β-solenoid.95-98

240

paired and intertwined protofilments.97 PrPC has no resistance to proteinase K (PK) while PrPSc

241

has some resistance.28 The difference in resistance to PK is presumed to be due to the β-sheet

242

secondary structure. Detecting and distinguishing among protein conformations is challenging.99

243

Although resistance to PK digestion is associated with PrPSc there is considerable

244

variation among the different strains and even among the different sizes of multimers in a given

245

prion strain.103-104 Prions can be differentially sedimented by ultracentrifugation into populations

246

of smaller multimers that are PK sensitive and larger multimers that are PK resistant.105 Even

247

though one fraction is PK resistant and the other fraction is PK sensitive, both are infectious and

248

possess the same primary, secondary, and tertiary structures.106

249

monomers present in the PrPSc multimer, resistance to PK is thought to be related to the presence

250

of β-sheet secondary structure.95, 98

251

adapted scrapie, are much more sensitive to PK than are other hamster-adapted strains, even

These β-solenoids are stacked and interlocked to form a quaternary structure of

In addition to the number of

Some prion strains, such as the drowsy strain of hamster-



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though each strain is composed of the same PrPC monomers, albeit in different conformations.107-

253

108

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strains.71 The same is true of the prions that cause chronic wasting disease in elk.71 This means

255

that PK resistance is not always a reliable means of distinguishing between PrPSc and PrPC.

The prions associated with scrapie are more sensitive to PK digestion than are other prion

256

The normal cellular prion protein (PrPC) is a glycolipoprotein. It possesses a single

257

disulfide bond. It has two sites that are variably glycosylated with large glycans, so the protein is

258

a mixture of diglycosylated, monoglycosylated, and unglycosylated proteins.109-111 There is

259

considerable variation in the composition of the sugars comprising the glycans.111 The protein

260

has a glycosylphosphatidylinositol (GPI) anchor.112 The sugar composition of the GPI anchor

261

also varies.113-114 Although the protein sequence remains constant, the variations in the sugar

262

composition of the glycans and the saccharide portion of the GPI anchor means that

263

comparatively few of the PrPC molecules have the same molecular weight, which complicates a

264

mass spectrometry-based analysis of the whole prion protein.

265

Prion nomenclature can be confusing. In this review, PrP refers to the prion protein, PrPSc refers to the infectious

266

regardless of conformation, glycosylation or GPI anchor.

267

conformation or isoform. PrPC refers to the natively expressed normal cellular prion protein

268

conformation, which is not infectious. Recombinant PrP (rPrP) is expressed in E. coli, so it has

269

no glycosylation or GPI anchor, but does possess a disulfide bond.

270

conformation as PrPC. Regardless of name, all prion proteins (PrPC, PrPSc, rPrP, and PrP) have

271

the same protein sequence and possess a single disulfide bond.

rPrP has the same

272

Unlike the natively expressed monomeric PrPC, prions are multimeric.103 This means that

273

the PrPSc multimeric complex needs to be denatured before it can be analyzed by mass

274

spectrometry. Denaturing in concentrated guanidine hydrochloride (GuHCl) is an effective


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method of inactivating prions.115 Methanol precipitation of these solutions removes the GuHCl

276

which would interfere with subsequent analysis and concentrates the protein contained in the

277

sample.38 In this way prions can be analyzed without contaminating instruments.

278

Prion proteins are irregularly glycosylated with glycans of varying composition, so

279

simple analysis of the whole protein is intractable.109-111 Instead the inactivated PrPSc can be

280

reduced, alkylated, and then digested with trypsin to yield a set of characteristic tryptic or

281

chymotryptic peptides suitable for MRM-based analysis.116-117 The required internal standards

282

can be conveniently generated from recombinant

283

once in the prion protein, so peptides can be used to demonstrate the presence of PrPSc and

284

quantitate the amount of PrPSc present in the sample.38,

285

permits researchers to quantitate covalent modifications of PrPSc, such as methionine oxidation

286

(Figure 6).71,

287

polymorphisms present in PrPSc from heterozygous sheep naturally infected with classical or

288

atypical scrapie.65, 119 MRM permits the straightforward detection and quantitation of a protein

289

that otherwise would be extremely difficult.

118

15

N-labeled PrP.71 Each peptide occurs only

116-117

Such peptide-based analysis

This approach was used to quantitate the relative proportion of PrP

290

Prions are known to infect and thereby contaminate the tissues of variety of food animals.

291

The prototypical prion disease is scrapie in sheep.81 The classical form of the disease was

292

described in the 18th century and perhaps even earlier.82 It is readily transmitted among sheep

293

and goats. A more recent form of scrapie, atypical scrapie, was first described in the late 20th

294

century.120 Its epidemiology is consistent with a sporadic origin.121 Based on analysis of human

295

consumption of sheep, there is no evidence that scrapie is transmissible to humans (zoonotic).122-

296

123

297

moose).124 It was first identified as a prion disease in 1978 and there is no epidemiological

Chronic wasting disease (CWD) is a prion disease of wild and farmed cervids (deer, elk



298

evidence that it is zoonotic.125 BSE transmitted to domestic cattle by the consumption of BSE

299

contaminated feed is referred to as classical BSE.126 The atypical forms of BSE (H-type or L-

300

type) are consistent with a very rare sporadic origin. The human manifestation of BSE is variant

301

Creutzfeldt-Jakob disease (vCJD), which is distinguishable from CJD.80

302

Different prions or strains, having characteristic phenotypes, are formed from the same

303

refolded PrPC.127 Furthermore, these prion strains replicate their properties in a given host.128

304

For example, if a sheep is infected with a BSE prion, the resulting prions are potentially zoonotic

305

even though they propagate in a sheep, whereas scrapie prions propagated in a sheep are not

306

zoonotic, despite the sheep expressing the same PrPC sequence as the one infected with BSE.128

307

Classical BSE (cBSE) is transmitted to cattle by consumption of BSE-contaminated feed, while

308

atypical BSE is thought to be a spontaneous disease.129-130 Sheep can be infected with classical

309

scrapie, which is acquired by transmission from a scrapie-infected sheep or by contact with a

310

scrapie-contaminated environment.81 The atypical form of scrapie, like BSE, is thought be a rare

311

spontaneous disease.121 There are varieties or strains of prions, and each can replicate its own

312

phenotype, which means that prions have the capacity to respond to selection pressures, i.e., they

313

can evolve.

314

among the prion strains.

The challenge of prion detection is to not only detect prions, but to distinguish

315 316

Detecting prions using mass spectrometry

317 318

Prions have been extensively analyzed by mass spectrometry.131 As noted previously the

319

work has been performed on trypsin or chymotrypsin digests of PrP. 65, 99, 117, 119, 132 The earliest

320

work demonstrated that the post-translational modifications present in PrPSc were also present in


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PrPC.71, 89-90 Later, mass spectrometry was used to verify that the composition of the asparagine

322

bound glycans and the GPI anchor were similarly varied in both PrPC and PrPSc. The presence

323

of significant amounts of β-sheet is presumed to impede PK digestion.95, 98 Mass spectrometry

324

has been used to establish the boundaries of the β-sheet structure by determining the identity of

325

the truncated peptides remaining after a partial PK digestion. It has been used to determine the

326

relative susceptibility of the two polymorphisms present in a heterozygous sheep naturally

327

infected with scrapie or the sporadic atypical scrapie. Mass spectrometry has been used to detect

328

prions and diagnose prion diseases without the need to digest the sample with PK.

329

The conformational changes that occur when PrPC adopts the PrPSc conformation can

330

dramatically change the chemical environment of an amino acid contained within the protein.95,

331

98

332

on the PrP conformation. In principle such a difference in reactivity would result in a covalent

333

difference that would remain after PrPSc had been denatured to PrP prior to analysis (Figure 7).

334

This conformation dependent difference in reactivity is observed in a lysine that is part of the

335

epitope of the 3F4 or the 6D11 monoclonal antibodies (mAb).88 In the PrPC conformation, the

336

lysine in the epitope is exposed. In the PrPSc conformation it is a cryptotope or hidden epitope.

337

It has been used as part of a conformation dependent immunoassay (CDI) that measures the

338

difference in signal intensity from denatured and non-denatured samples.104 The ε-amino group

339

of this lysine also reacts readily with N-hydroxysuccinimide esters of carboxylic acids to form

340

the corresponding ε-amide. When the ε-amino group of this lysine is amidated, it is no longer

341

recognized by the 3F4 or 6D11 mAbs. The manipulations necessary for these reactions adds 30

342

minutes to the analysis time.

343

additional structural information it generates. In this way a mAb can be used to measure the

This means that the same amino acid can react differently with the same reagent, depending

This modest addition is more than compensated for by the



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344

amount PrPSc in a sample without using PK and in the presence of PrPC.88 Mass spectrometry

345

was used to observe the reaction of acetic anhydride or tetranitromethane with PrPSc.133

346

Mass spectrometry has been used to distinguish among prion strains.99 Often prion

347

strains differ in terms of their PK cleavage sites. Mass spectrometry has been used to identify

348

these sites and thereby distinguish between scrapie strains in sheep. Another approach exploits

349

the reactivity of the ε-amino group of lysine with the N-hydroxysuccinimide ester of acetic acid

350

(Ac-NHS). In this example, four of the five prion strains have identical PK cleavage products

351

(Figure 8).99 The strains were reacted with fixed concentrations of Ac-NHS for a fixed time.

352

The samples were not digested with PK and MRM was used to monitor the extent of acetylation

353

for the various lysines in PrP (Figure 9). This was necessary, since the appropriate mAbs to

354

detect all lysines in PrP do not exist. The extent of acetylation was different for each of the five

355

prions examined, which is consistent with each strain having a characteristic conformation.99

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Mass spectrometry can be used to both detect prions and to distinguish among prion strains.

357

Fortunately, the one zoonotic prion disease, BSE, is rapidly disappearing. In 2016 there

358

were no cases of classical BSE identified in the United Kingdom, the place where BSE was first

359

identified and where the largest number of cases was diagnosed. 134 It is likely that classical BSE

360

soon will cease to exist.

361

Although CWD and scrapie have not been shown to be zoonotic,

122-123

there is some

362

experimental evidence to suggest they have that potential. The zoonotic potential of CPD

363

(camels) is not currently known, but will be defined by future work.87 Transgenic mice were

364

used to show that some scrapie strains have zoonotic potential.135 Scrapie was successfully

365

transmitted to cynomolgus macaques, a relevant human model, suggesting the potential

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zoonosis.136 Squirrel monkeys succumbed to a prion disease after infection with CWD.


137-138

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Analogous attempts to infect cynomolgus macaques with CWD failed, suggesting that the tested

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strains of CWD are not zoonotic.

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CWD strains may be transmissible to cynomolgus macaques.140

370

appears that the need to monitor prions and prion strains will remain as long as prions exist.

371

Since mass spectrometry can directly detect prions and distinguish among prion strains without

372

using PK,65, 99, 117, 119 it will remain an invaluable analytical tool for the food forensicist.

373

137-139

Preliminary work in Canada suggests that Canadian Even though BSE is waning, it

There are other proteins that form amyloids and are found in food.141-142 Foie gras

374

contains amyloid.

Researchers have demonstrated that this amyloid can be transmitted to

375

vulnerable mice by oral transmission or by intravenous injection. It is not clear if the mouse

376

model applies to humans, but it does suggest the possibility that other protein refolding diseases

377

may also be transmissible by food.

378

provide a direct means of identifying protein conformations.

Fortunately for food forensicists, mass spectrometry may

379 380

Shiga toxins143

381 382

Shiga toxin was originally described, in the 19th century, as a toxin produced by the

383

bacterium Shigella dysenteriae.144 Much later, a similar toxin was isolated from cultures of E.

384

coli and referred to as type 1 Shiga toxin.44,

385

structurally and functionally related toxins, from other cultures of E. coli, that are referred to as

386

type 2 Shiga toxins (Figure 10).

387

verotoxins and Shiga-like toxins. Now they are all referred to as Shiga toxins.146 They are

388

classified into two types of Shiga toxins, type 1 and type 2, based on their amino acid sequence.

389

Both toxins are AB5 hexamers, in which the B subunits bind to a target eukaryotic cell and the A

145

Other researchers identified different, but

At various times these toxins have been referred to as



390

subunit contains a catalytic domain that causes the death of the eukaryotic host cell.147 Both

391

types of toxins can cause serious human disease, but type 2 Shiga toxins are most often

392

associated with the more serious symptoms of Shiga intoxication.

393

The Shiga toxins produced by Shiga toxin-producing E. coli (STEC) are the major

394

virulence factor associated with these food-borne illnesses.149 Shiga toxins are hexameric (AB5)

395

proteins (~70kDa) composed of an A subunit (~32 kDa), which contains the catalytic domain,

396

and is non-covalently bound to five identical B subunits (~7-8 kDa), which bind to specific

397

glycolipids on the surface of target eukaryotic cells.150 The B subunits bind to the saccharide

398

portion of the glycolipids globotriaosylceramide (Gb3) and/or globotetraosylceramide (Gb4).151-

399

155

400

binds to the Gb3 and/or Gb4 glycolipids on a detergent resistant portion of a membrane, then the

401

toxin can enter the cell. This binding of the toxin to a cell surface determines which cells are

402

affected by the toxin.

Mutations in the B subunits can alter the binding specificity of the B subunits.156 If the toxin

403

The binding of the toxin to a eukaryotic cell surface induces the endocytosis of the Shiga

404

toxin into the cell.157 The endocytosed toxins are sorted for retrograde trafficking instead of

405

lysosomal digestion. Retrograde trafficking moves the toxin through the Golgi apparatus to the

406

endoplasmic reticulum. During this process the A subunit is cleaved by a host enzyme (furin)

407

and a disulfide bond is cleaved to free the catalytic domain of the toxin (A1). The A1 domain

408

contains an N-terminal signal sequence which facilitates its retrotranslocation from the

409

endoplasmic reticulum to the cytosol. Once in the cytosol, the A1 domain is free to cleave a

410

specific adenine (A4324 in rat) of the 28S rRNA component of the ribosome, thereby halting

411

protein synthesis.158-159 A single functioning A1 subunit is sufficient to kill a eukaryotic cell

412

(Figure 11).


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A sixteen-year survey (1998-2014) determined that while STEC was associated with only

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2.5% of foodborne illness, it accounted for 13% of hospitalizations, and nearly 11% of the deaths

416

associated with foodborne illness.160 Every year approximately 175,000 Americans are infected

417

with STEC.15 Most patients (83%) have comparatively mild symptoms, such as severe stomach

418

cramps and sometimes diarrhea, but they are not serious enough to require hospitalization.161

419

There is often no fever or only a mild one (

Food Forensics: Using Mass Spectrometry To ... - ACS Publications

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