Species Differentiation and Quantification of Processed Animal

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Species Differentiation and Quantification of Processed Animal Proteins and Blood Products in Fish Feed using an 8-plex Mass Spectrometry-Based Immunoassay Andreas E. Steinhilber, Felix F. Schmidt, Wael Naboulsi, Hannes Planatscher, Alicia Niedzwiecka, Jutta Zagon, Albert Braeuning, Alfonso Lampen, Thomas O. Joos, and Oliver Poetz J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03934 • Publication Date (Web): 17 Sep 2018 Downloaded from http://pubs.acs.org on September 18, 2018

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Journal of Agricultural and Food Chemistry

Article

Species Differentiation and Quantification of Processed Animal Proteins and Blood Products in Fish Feed using an 8-plex Mass Spectrometry-Based Immunoassay Andreas E. Steinhilber3, Felix F. Schmidt1, Wael Naboulsi3, Hannes Planatscher3, Alicia Niedzwiecka2, Jutta Zagon2, Albert Braeuning2, Alfonso Lampen2, Thomas O. Joos1, and Oliver Poetz*1,3

1

NMI Natural and Medical Sciences Institute at the University of Tuebingen, 72770

Reutlingen, Germany 2

German Federal Institute for Risk Assessment, Dept. Food Safety, 10589 Berlin, Germany

3

SIGNATOPE GmbH, 72770 Reutlingen, Germany

1

ACS Paragon Plus Environment


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1

ABSTRACT

2

With the reintroduction of non-ruminant processed animal proteins (PAPs) for the use in aquaculture in

3

2013, there is a suitable alternative to replace expensive fish meal in fish feed. Nevertheless, since the

4

bovine spongiform encephalopathy (BSE) crisis, the use of PAPs in feed is strictly regulated. To date,

5

light microscopy and polymerase chain reaction are the official methods for proving the absence of

6

illegal PAPs in feed. Due to their limitations, alternative methods for the quantitative species

7

differentiation are needed. To address this issue, we developed and validated an 8-plex mass

8

spectrometry-based immunoassay. The workflow comprises a tryptic digestion of PAPs and blood

9

products in suspension, a cross-species immunoaffinity enrichment of 8 species-specific alpha-2-

10

macroglobulin peptides using a group-specific antibody and a subsequent analysis by ultra-high-

11

performance liquid chromatography coupled to tandem mass spectrometry for species identification

12

and quantification. This workflow can be used to quantitatively determine the species origin in future

13

feed authentication studies.

14 15

KEYWORDS

16

tandem mass spectrometry; immunoaffinity enrichment; processed animal proteins; blood meal; meat

17

and bone meal; spray-dried plasma; species differentiation; feed authentication

18

2


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INTRODUCTION

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The expansion of aquaculture, the world’s fastest growing food production sector with an average

21

growth rate of 8-10% per year since 1970, was accompanied by a rapid growth of fish feed production

22

1

23

pushed to historic heights in late 2014 2. Consequently, sustainable ingredient alternatives to substitute

24

expensive fish oil and fish meal gained attention 3. However, these mainly plant-derived products

25

possess characteristics that make them less suitable ingredients than fish oil and fish meal 4. Another

26

opportunity came up in 2013 when the European Union reintroduced non-ruminant processed animal

27

proteins (PAPs) for the use in aquaculture by Commission Regulation (EU) No. 56/2013 5. Mammalian

28

PAPs are considered as a good alternative to replace expensive fish meal in fish feed 6.

. The prices for fish oil and fish meal, the most nutritious and digestible ingredients in fish feed, were

29

PAPs are valuable feed ingredients that increase the overall sustainability of the food chain, since

30

they are produced from animal by-products that are not intended for human consumption. PAPs are

31

known to improve the nutritional value of feed for monogastric animals like chicken or pig, since they

32

provide high levels of fat, proteins, minerals and even essential vitamins compared to other feed

33

ingredients 6-8. In aquaculture, the suitable use of poultry PAPs in feed for salmonids has already been

34

reported

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products such as spray-dried hemoglobin meal (SDHM) or spray-dried plasma (SDP) in the sense of

36

the Regulation (EU) No. 142/2011 11 are two types of animal feed additives that are gaining popularity

37

due to their high digestibility

38

proteins is strictly regulated as a consequence of the bovine encephalopathy (BSE) crisis (Table S1). A

39

risk assessment performed by the European Food Safety Authority (EFSA) was necessary to

40

reauthorize non-ruminant PAPs for the use in aquaculture feed 14. Ruminant PAPs are still prohibited

41

and therefore they are not allowed as substitutes for fish meal in fish feed.

9-10

. Blood meal (BM), representing a special category of PAP, and lower processed blood

12-13

. Nevertheless, in the European Union, the use of these animal

3



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To ensure feed safety, analytical methods have been implemented to prove the absence of illegal

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PAPs in feed. In 1998 optical light microscopy has been implemented as the first official method for

44

the detection of illegal additives (e.g. feathers, bones) 15. In visually not classifiable materials, such as

45

PAPs and blood products, light microscopy gives no clue about which species are present. As a

46

consequence, the polymerase chain reaction (PCR) that allows a reliable species identification based on

47

the analysis of DNA, was introduced as complementary and second tier method 16. However, the use of

48

PAPs in fish feed requires high processing conditions (133°C, 20 min 3 bar for mammalian PAPs) 11,

49

thus affecting the accuracy of DNA-based methods due to fragmentation reactions. Furthermore, PCR

50

is not able to determine the tissue origin of a sample and therefore cannot differentiate legal milk

51

additives from illegal ruminant PAPs 17. The limitations of the current analytical methods have driven

52

the development of alternative methods.

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Protein-based methods like immunoassays seemed to be a promising approach since they offer

54

species and tissue specificity as well as the possibility for quantification. The latter is important since

55

producers, represented by the European Fat Processors and Renderers Association (EFPRA), but also

56

control laboratories call for quantitative accurate thresholds to replace the pending zero-tolerance-

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concept of PAPs in feed. Proteins suffer from denaturation at high temperatures. For this reason,

58

immunoassays for the quantification of PAP samples are limited to the analysis of heat-stable proteins

59

18

60

or tryptic peptides 19. During the past decade, the mass spectrometric (MS) analysis of tryptic peptides has emerged as a 20

61

powerful tool in the field of proteomics

62

chemistry. MS methods for the species authentication of meat products have been extensively

63

described 21. One approach of species identification in meat products is untargeted MS combined with

64

spectral libraries. This approach was shown in both, raw and cooked samples, proving the suitability of

and has been quickly adopted in related fields like food

4


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22

65

peptide-centric MS for processed protein analysis

. Also, in the field of PAP authentication MS

66

methods receive a growing attention

67

differentiation of PAP samples

68

MS methods have to be developed 26-28. Targeted MS methods like selected reaction monitoring (SRM)

69

or multiple reaction monitoring (MRM) allow a highly specific and sensitive detection of peptides and

70

indirectly proteins. Targeting proteins from different tissue types allows a detection of peptides in a

71

species and tissue-specific way, independent from protein folding and therefore, enable the analysis of

72

PAPs. The differentiation of bovine PAPs and milk products by targeted MS was recently reported

73

30

74

signal to signals from spiked stable isotope labelled standard peptides.

23-25

. Recently, untargeted MS was introduced for the species

26

. However, when it comes to high-sensitive quantification, targeted

29-

. Quantification in targeted MS methods can be achieved by referencing the endogenous peptide

75

To further improve throughput and sensitivity, hybrid methods that combine immunoprecipitation

76

with liquid chromatography coupled to mass spectrometry (LC-MS), have been established on both

77

MALDI-MS and ESI-MS platforms

78

pharmaceutical research for the quantification of receptors 33, kinases 34, drug-metabolizing enzymes 35

79

and plasma proteins

80

specific quantification of banned ruminant PAPs in feed

81

homologous peptides from different species in a cross-species epitope approach using only one

82

antibody. Species identification is performed subsequently in an ultra-high-performance liquid

83

chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis using parallel reaction

84

monitoring (PRM) and stable isotope labelled standard peptides as internal standards for quantification.

85

The developed method can be used to quantitatively determine the species origin of PAPs and blood

86

products in future feed authentication studies.

31-32

. So far, immunoaffinity MS is used in clinical and

36

. Recently, we introduced immunoaffinity MS to feed analysis for the species37

. Here, we apply this concept to enrich

87 5



88

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MATERIALS AND METHODS

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Samples. Animal protein samples of different species and tissue origin were compiled by the

90

German Federal Institute for Risk Assessment (Berlin). In this study, two blood meal (BM) samples,

91

one porcine and one mix of poultry animals, one bovine meat and bone meal (MBM; treated at least

92

133 °C, 3 bar, 20 min), one bovine and two porcine spray-dried plasmas (SDP) provided by

93

NutriControl (Veghel, The Netherlands) were analyzed. One SDP sample of unknown species origin

94

was obtained from T.T. Baits (Erlangen, Germany). Furthermore, four porcine MBM (LUFA Nord-

95

West, Oldenburg, Germany) and one mix of poultry MBM (GePro, Diepholz, Germany) were studied.

96

As feed matrices, two fish feeds, one with and one without land living animals were used (BioMar,

97

Brande, Denmark). For assay validation we purchased native citrate plasmas of nine species (cattle,

98

sheep, goat, pig, horse, chicken, turkey, duck and goose) from preclinics GmbH (Potsdam, Germany).

99

Preparation of Validation Samples. To validate assay specificity, eight mixtures of digested citrate

100

plasmas in digested fish feed were prepared (10% w/w). In each mixture one of the nine species was

101

left out. Sheep and goat plasma were mixed in equal amounts and treated as one species since the target

102

peptide was the same in both species. To validate the assays intra- and inter-assay variation, mixtures

103

of all species in fish feed on the solid non-digested level were prepared. Native citrate plasmas of nine

104

species were mixed in equal volumes and lyophilized for two days using an alpha I-6 freeze dryer

105

(Christ, Osterode, Germany). The fine multispecies powder was then mixed in fish feed at three

106

concentrations (1%, 5%, 10% w/w). Consequently, the single species concentrations are 0.1%, 0.6%

107

and 1.1% (w/w), respectively. Before mixing, the coarse fish feed powder was further homogenized

108

into a fine powder using a ball mill (Sartorius, Goettingen, Germany). Therefore, 80 mg of fish feed

109

was weighed into cryovials, cooled in liquid nitrogen and ground to a fine powder using 7 mm steel

6


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balls at 2000 rpm for 2 min. The fine powders were then mixed at the three concentrations. To obtain a

111

homogeneous mixture, glass beads with a diameter of 2 mm were added and mixed properly.

112

Cross Species Epitope Identification. Cross species epitopes were identified via bioinformatics. 38

113

150 high abundant plasma proteins published by Anderson

were translated into gene names and the

114

respective protein sequences of all species of interest were fragmented in silico into tryptic peptides. To

115

minimize analytical issues, we filtered for peptide lengths between 8 and 25 amino acids and peptides

116

that do not contain cysteine and methionine. The remaining peptides were grouped based on their C-

117

terminal sequence comprising four amino acids. We prioritized the cross-species epitopes by their

118

species coverage.

119

Protein Determination. Plasma protein concentrations were determined with a Pierce bicinchoninic

120

acid assay kit (Thermo Fisher Scientific, Waltham, USA). Bovine serum albumin (BSA) was used as

121

the protein standard in concentrations from 25 µg/mL to 2 mg/mL in phosphate buffered saline (PBS).

122

Plasma samples were diluted 1:50 in PBS and 25 µL of sample dilution mixed with 200 µL of reagent

123

solution. After incubation for 30 min at 37 °C, absorption at 562 nm was measured. The plasma

124

concentrations were calculated using four parameters curve fitted to the BSA standards.

125

Total Peptide and Protein Content Estimation. The total protein and peptide amount in the

126

digested samples after heterogeneous phase digestion (HPD) was estimated by an UV absorption

127

measurement using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA).

128

Therefore, 2 µL of digested sample were placed on the Nanodrops sensor and the absorbance at 280 nm

129

was measured. Peptide amount were calculated by applying 1 Abs ≈ 1 mg/mL. UV absorbance due to

130

added trypsin and hydrogen iodide formed during alkylation with iodoacetamide was subtracted using a

131

blank digest.

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Heterogeneous Phase Digestion (HPD). 620 µL of triethanolamine digestion buffer (50 mM)

133

containing 0.5% n-octylglucoside (NOG) was added to 15 mg of animal protein or fish feed. The

134

suspension was heated 5 min at 99 °C and cooled down to room temperature. The sample was reduced

135

with tris(2-carboxyethyl)phosphine (TCEP) at a final concentration of 5 mM for 5 min at room

136

temperature. Iodoacetamide (IAA) was added to a final concentration of 10 mM and the samples were

137

alkylated for 30 min at room temperature in the dark. Trypsin (Worthington, Lakewood, USA) was

138

added in a 1:40 ratio based on the initial sample weight. The samples were digested at 37 °C for 2 h

139

while shaking at 1000 rpm to achieve a stable suspension. The digestion was stopped by adding

140

phenylmethanesulfonyl fluoride (PMSF) at a final concentration of 1 mM. The suspension was

141

centrifuged 5 min at 13000 rcf. Afterwards, the supernatant was transferred to a new reaction tube. The

142

supernatant’s peptide content after digestion was estimated by UV spectroscopy as described in the

143

previous section.

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Extraction and In-solution Digestion (ISD). Protein extraction with subsequent tryptic digestion in

145

solution was performed as described in the HPD section. However, instead of adding trypsin, the

146

sample was incubated under the same conditions without enzyme. The insoluble fraction was removed

147

by centrifugation and the supernatant was digested by trypsin for 2 h at 37 °C. The digestion was

148

stopped by adding PMSF at a final concentration of 1 mM. The protein content estimation was

149

performed via UV spectroscopy.

150

Digestion of Citrate Plasma. 1.5 mg of native citrate plasma was diluted to a final volume of

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620 µL in triethanolamine digestion buffer (50 mM) containing 0.5% n-octylglucoside (NOG).

152

Denaturation, alkylation and digestion by trypsin were performed as described in the HPD section;

153

however, the solution was shaken at 650 rpm. The digestion was stopped by adding PMSF at a final

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concentration of 1 mM. The digest’s final volume was 750 µL and therefore the protein concentration

155

was assumed to be 2 mg/mL.

156

Immunoprecipitation. Different known amounts of enzymatically fragmented samples were

157

incubated with 1 µg antibody. The antibody was generated according to a protocol we reported earlier

158

39

159

50 fmol. C-terminal lysine or arginine were

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samples were incubated 1 hour at room temperature and the peptide antibody complexes were

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precipitated with 5 µL protein G-coated magnetic microspheres (Thermo Fisher Scientific, Waltham,

162

USA). The conjugates were washed two times in 100 µL phosphate buffered saline and three times in

163

100 µL of 50 mM ammonium bicarbonate, each containing 0.03% (w/v) CHAPS as a detergent.

164

Peptides were eluted in 20 µL 1% formic acid (FA).

. For quantification, stable isotope labelled standard peptides were added at constant amount of 13

C/15N-labelled (Intavis AG, Tuebingen, Germany). The

165

Chromatography. Chromatographic separation of peptides was performed on a nanoliter flow

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UHPLC system (Ultimate 3000 RSLCnano, Thermo Fisher Scientific). 5 µL of the eluted peptides

167

were loaded on an Acclaim PepMap100 C18 µ-pre-column (0.3 mm I.D. x 5 mm, 5 µm, Thermo Fisher

168

Scientific) for 0.25 min at a flow rate of 120 µL/min in LC-MS grade water containing 2% ACN and

169

0.05% TFA. The peptides were separated in 5 min by an Acclaim PepMap RSLC C18 (75 µm I.D. x

170

150 mm, 2 µm, Thermo Fisher Scientific) using a gradient from 4% to 15% organic phase in 0.5 min

171

and 15% to 35% organic phase in 4.5 min at a flow rate of 1 µL/min and a column temperature of

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55 °C. The column was washed and equilibrated for the next run in another 5 min. Aqueous phase

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consisted of 0.1% FA in LC-MS grade water. The organic phase consisted of 80% ACN and 20% LC-

174

MS grade water containing 0.1% FA. The same system was used for identification experiments in

175

complex digests. Here a linear gradient from 5% to 55% in 180 min was used.

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Mass Spectrometry. The UHPLC system was coupled to a QExactive Plus hybrid quadrupole

177

orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, USA). For targeted data acquisition a

178

PRM method was applied. The resolution was set to 35 000, the AGC target of 2E5 and the maximum

179

injection time to 60 ms. Precursor m/z are supplied by an inclusion list and isolated with a mass

180

window of 1.5 m/z. Normalized collision energy was set to 25. Spectral multiplexing was set to 2 for

181

light and heavy peptide pairs. Skyline v3.7 was used for data analysis and the three most intense

182

fragment ions of each target peptide were integrated. Identification experiments were performed on the

183

same system applying a top 10 Full MS/ddMS2. Full MS resolution was set to 70 000 with an AGC

184

target of 3E6 and a maximum injection time of 100 ms. The scan range was set to 300 to 2000 m/z.

185

Data dependent MS2 spectra were acquired with a resolution of 17 500 and an AGC target of 5E5 with

186

a maximum injection time of 50 ms. The isolation window of precursor ions was set to 2.0 m/z.

187

Normalized collision energy was set to 25. Dynamic exclusion of precursors was set to 5.0 s.

188 189

RESULTS AND DISCUSSION

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Cross Species Epitope Selection. Five plasma proteins that allow a cross-species enrichment of

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homologous peptides using only one antibody were bioinformatically identified: alpha-2-

192

Macroglobulin, coagulation factor VIII, antithrombin-III, serum albumin and cholinesterase (Table 1).

193

The cross-species epitope from alpha-2-macroglobulin (A2M) was chosen to generate a polyclonal

194

antibody. Cholinesterase was not chosen, because of its lower plasma abundancy compared to the other

195

four targets. Serum albumin was not chosen because of the high risk for cross contaminations in

196

biochemistry laboratories since it is used as blocking reagent and standard protein in immunoassays

197

and protein estimation assays. The antithrombin peptides comprised a very long epitope at the C

198

terminus which would allow the generation of a highly specific cross-species antibody. However, the 10


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peptides are very large with quite similar epitope sequences and therefore a chromatographic separation

200

was supposed to be challenging. Factor VIII comprised shorter peptides with a higher inter-species

201

sequence variation. The short cross-species epitope of these target peptides comprises only four amino

202

acids which increases the risk for antibody cross-reactions to other high abundant sequences. In

203

comparison, A2M is superior to the other target proteins: First, the C-terminus covers nine species of

204

interest. A differentiation of sheep and goat via A2M is not possible, but this is not an issue since the

205

legal regulations state ruminants as one group. Second, the N-terminal sequences show a high inter

206

species variability which facilitates a chromatographic separation. Third and most important, the

207

conserved C-terminal sequence offers the possibility of expanding the cross-species epitope to a length

208

of eight amino acids if an immunization with the two sequence variations, containing either

209

phenylalanine (F) or tyrosine (Y) in the X position of the epitopes sequence (VEEXVLPK) is

210

performed. This allows the generation of a cross-species antibody which is capable of enriching all

211

peptides with a high specificity.

212

Sample Preparation. PAP and blood product samples were prepared for LC-MS/MS analysis using

213

a protocol we validated and published recently 37: Protein extraction and digestion were performed in a

214

single step using heterogeneous phase digestion (HPD). For a porcine blood meal and a porcine spray-

215

dried plasma sample, the HPD protocol was compared with an extraction in the same buffer and

216

subsequent In-solution Digestion (ISD) of the supernatant. An absorption measurement at 280 nm

217

indicated that HPD released more peptides compared to ISD as it was seen in the previous study for a

218

bovine meat and bone meal sample 37. Peptide amounts released by HPD were nearly two times higher

219

in BM and five times higher in SDP (data not shown). The developed 8-plex MS-based immunoassay

220

was applied to the same protein amounts after ISD and HPD, respectively. Here it was seen that not

221

only the total peptide amount was higher, but also the porcine A2M peptide was released in higher 11



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amounts using HPD (Figure S1). The peptide release increased by nearly the threefold for BM and

223

tenfold for SDP. Thus, the compatibility of the recently developed HPD protocol with BM and SDP

224

samples was confirmed.

225

Digestion Kinetics. For a highly sensitive quantification of proteins by the indirect analysis of

226

peptides, the digestion step is crucial. It was observed that a tryptic digestion is strongly impacted by

227

the digestion time

228

others were rapidly released and then showed a decreasing concentration with further digestion. In case

229

of HPD, where the digestion takes place at the liquid-solid-interface, the analysis of the digestion

230

kinetics is also very important in order to achieve the maximum peptide release. However, PAP

231

samples were not available for all species. For this reason, the time dependent study was performed

232

with each species’ citrate plasma (Figure S2). Mostly, the peptides showed a relative constant

233

appearance during tryptic digestion up to 42 h. In contrast, the A2M-specific peptides from turkey and

234

goose showed steadily decreasing concentrations after 2 h digestion time. The chicken-specific peptide

235

showed a constant concentration up to 10 h and rapid degradation at overnight digestion (16 h) and

236

longer digestion times. The highest relative mean peptide release was observed to be at 2 h which was

237

then chosen as the standard digestion time. Since different sample types could affect HPD, the

238

digestion kinetics were analyzed in three different bovine protein samples: citrate plasma, meat and

239

bone meal (rMBM) and spray-dried plasma (rSDP) (Figure S3). Native citrate plasma and rSDP

240

showed very similar peptide releases. The peptide release from rMBM was slightly different at a

241

digestion time of 16 h, however, the overall trend was the same including the rapid degradation after

242

24 h to 42 h. The application of HPD to different sample types was considered to be unproblematic.

35, 40

. Some target peptides showed a slow release during tryptic digestion while

243

Linearity and Sensitivity in Fish Feed. The assay’s linearity and sensitivity was tested by

244

analyzing a dilution series of the light peptides (0.05 – 1000 fmol) at a constant amount of isotope 12


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labelled standard peptides (50 fmol) in digested fish feed as matrix (100 µg) using the generated anti-

246

VEEXVLPK antibody (1 µg). The mass spectrometric detection after immunoaffinity enrichment and

247

chromatographic separation was performed using a high resolution and accurate mass (HRAM)

248

quadrupole-orbitrap hybrid mass spectrometer. Compared to low resolution triple quadrupole

249

instruments that are common in routine analysis it is possible to measure both, precursor and fragment

250

ions with an outstanding accuracy and sensitivity

251

selected ion monitoring (SIM), based on precursor analysis and (ii) parallel reaction monitoring (PRM),

252

based on fragment ion analysis after higher energy collision induced dissociation (HCD).

41-42

. Basically, there are two targeted modes: (i)

253

The measured signal ratios plotted against the concentration ratios of light and heavy peptides

254

showed a linear relationship over a concentration range of 3 to 4 orders of magnitude (Figure 1). The

255

coefficients of determination (R²) ranged between 0.9908 and 0.9999 using PRM and between 0.9972

256

and 0.9999 using SIM detection (Table 2). Only signals above the determined LOD were used to assess

257

the linearity. The limit of detection (LOD) was determined by a method validated earlier by Mani and

258

colleagues

259

based peptide assays. The determination is based on the measurement of blank samples as well as low

260

concentrated analyte samples, considering alpha and beta errors. The LODs observed in PRM were

261

consistently lower compared to SIM. The detection sensitivity using PRM ranged between 30 amol

262

(cattle) and 693 amol (pig). In SIM the LODs ranged between 248 amol (duck) and 12.9 fmol (goose).

263

The higher specificity in PRM detection mode leads to reduced noise levels and therefore lower

264

concentrations can be detected. Moreover, the confidence of peptide identification due to the fragment

265

ion pattern is higher in PRM, for which reason it was chosen as detection method in all further

266

experiments.

43

. This method was shown to reliably determine LOD and LOQ in mass spectrometry-

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The LODs mentioned above are related to the absolute peptide amounts in the immunoaffinity

268

enrichment step. In feed analysis the LODs are commonly expressed as weight percentages (% w/w) of

269

animal protein in a feed compound. Since the achievable sensitivity mainly depends on the extracted

270

peptide amount from highly processed protein samples, two meat and bone meals from the species pig

271

(pMBM) and cattle (rMBM) were analyzed in different concentrations in fish feed (Figure 2). The two

272

MBMs and the fish feed (FF) were separately digested using HPD and a dilution series of the two

273

MBMs in FF was prepared. A total amount of 100 µg sample mix was analyzed with the 8-plex MS-

274

based immunoassay. The lowest concentration that could be detected above the LOD was 0.1% (w/w)

275

corresponding to an absolute amount of 19.2 amol for rMBM and 0.25% (w/w) corresponding to an

276

absolute amount of 1.56 fmol for pMBM. These LOD results are consistent with the results that were

277

observed in the standard peptide dilution experiment (Table 2). A quantitative detection with

278

coefficients of variation of less than 20% was possible at 0.75% (w/w) for both rMBM and pMBM.

279

This experiment proofs the suitability of the 8-plex MS-based immunoassay for the detection and

280

quantification of PAPs in fish feed in the range of 0.1 – 0.75% (w/w).

281

Species Specificity. The species specificity of the multiplex MS-based immunoassay was

282

determined by analyzing nine citrate plasma mixtures, each missing one species. A specific multiplex

283

detection should be able to detect eight species while the missing species should not be detected. At

284

least three fragment ions of each precursor peptide and a coefficient of variation ≤ 20% were the

285

criteria for a positive detection. The expectations were fulfilled with one exception (Table 3). The fish

286

feed matrix that was not supposed contain land living animals, showed a slight amount of porcine

287

A2M. In the sample without porcine plasma while containing all other species, the signal for the A2M-

288

peptide was not significantly higher than that of the matrix itself (p = 0.67). This indicated a porcine

289

contamination of the fish feed and a pig-specific detection was assumed. 14


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Additionally, PAP and blood product samples were analyzed to check a species-specific detection in

291

processed samples (Table 4). The expected species origin was mostly confirmed by applying the 8-plex

292

MS-based immunoassay. Again, the fish feed without land living animals showed a slight porcine

293

contamination. In the second fish feed, which was supposed to contain land living animals, porcine

294

material and a slight amount of chicken material was detected. The unknown MBM and BM samples of

295

poultry mixtures were proven to be pure chicken in case of MBM and a mixture of 80% chicken and

296

20% turkey material in case of the BM sample. The ruminant MBM and SDP samples were confirmed

297

to be pure samples. Four porcine PAP samples of different origin, a porcine blood meal and two

298

porcine spray-dried plasmas were confirmed to be pure porcine samples. The SDP sample of unknown

299

species origin, was tested to consist of 80% bovine and 20% porcine material. In conclusion, the

300

analyzed clean citrate plasmas and the real samples proved the highly specific species differentiation in

301

PAPs and blood products after cross-species immunoaffinity enrichment and LC-MS/MS analysis

302

using PRM.

303

Intra- and Inter-Assay Precision. To assess intra- and inter-assay variance, three concentrations of

304

a plasma mixture in fish feed (1%, 5% and 10%, w/w) on the dried and non-digested level were

305

prepared and enzymatically fragmented by HPD. Single species concentrations in the fish feed were

306

0.1%, 0.6% and 1.1%, respectively. The 8-plex MS-based immunoassay was capable to measure all

307

peptides with coefficients of variation below 20% and at least three detected transitions for each target

308

peptide (Table 5). The assay including the HPD preparation is able to quantify species-specific peptides

309

with high intra- and inter-assay precision on a level of only 0.1% (w/w) in a feed matrix.

310

To conclude, in the present work we propose a workflow comprising a two-hour tryptic digestion of

311

denatured PAPs and blood products in suspension, followed by a cross-species immunoaffinity

312

enrichment using a group-specific antibody and a mass spectrometric analysis for the identification and 15



Page 16 of 26

313

quantification of species-specific peptides in a concentration range of 0.1 – 0.75% (w/w) in fish feed.

314

The new HPD protocol was proven to release more target peptides from blood meal and spray-dried

315

plasma samples compared to a protein extraction with tryptic digestion of the supernatant. The

316

immunoaffinity enrichment step allowed short chromatographic gradients with a cycle time of 10 min

317

and therefore an increased sample throughput. A time-consuming sample clean up before LC-MS/MS

318

analysis was not necessary. This 8-plex MS-based immunoassay was partially validated for proving

319

fish feed authenticity. However, this assay could be also used for farmed animals feed authentication

320

and other types of animal feed. Multiplex MS-based immunoassays are a promising tool to overcome

321

current limitations in feed authentication and meet the future requirements for quantitative official

322

methods in feed analysis.

323 324

ABBREVIATIONS

325

PAP, processed animal protein; MBM, meat and bone meal; SDP, spray-dried plasma; SDHM, spray-

326

dried hemo-globin meal; BM, blood meal; MRM, multiple reaction monitoring; SRM, selected reaction

327

monitoring; PRM, parallel reaction monitoring; LC, liquid chromatography; MS, mass spectrometry;

328

HPD, heterogeneous phase diges-tion; ISD, in-solution digestion; FF, fish feed

329 330

ACKNOWLEDGEMENT

331

A.E.S. and O.P. were supported by a grant of the Federal Ministry of Food and Agriculture (FKZ

332

28165035-14).

333 334

SUPPORTING INFORMATION DESCRIPTION

335

The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX 16


Page 17 of 26


336

Additional table describing the current legislation for the use of animal proteins as additives in feed,

337

table containing information about the m/z of precursor and fragment ions for the detection of alpha-2-

338

macroglobulin peptides; figures showing the improvement of HPD for sample preparation and the time

339

dependent peptide release.

340 341

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465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485

38.

486

FIGURE CAPTIONS

487

Figure 1. Linear range of the 8-plex MS-based immunoassay for each species-specific peptide

488

determined in 100 µg fish feed (FF) as matrix: A = cattle, B = horse, C = pig, D = sheep/goat, E =

489

chicken, F = duck, G = goose, H = turkey. Linearity is plotted as measured signal ratio over actual

490

concentration ratio of analyte to internal standard (IS). The limit of detection (LOD) is shown as

491

dashed horizontal line (n=3).

39.

40.

41.

42.

43.

Hortin, G. L.; Sviridov, D.; Anderson, N. L., High-abundance polypeptides of the human plasma proteome comprising the top 4 logs of polypeptide abundance. Clinical chemistry 2008, 54 (10), 1608-16. Hoeppe, S.; Schreiber, T. D.; Planatscher, H.; Zell, A.; Templin, M. F.; Stoll, D.; Joos, T. O.; Poetz, O., Targeting peptide termini, a novel immunoaffinity approach to reduce complexity in mass spectrometric protein identification. Mol Cell Proteomics 2011, 10 (2), M110 002857. Proc, J. L.; Kuzyk, M. A.; Hardie, D. B.; Yang, J.; Smith, D. S.; Jackson, A. M.; Parker, C. E.; Borchers, C. H., A quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin. J Proteome Res 2010, 9 (10), 5422-37. Michalski, A.; Damoc, E.; Hauschild, J. P.; Lange, O.; Wieghaus, A.; Makarov, A.; Nagaraj, N.; Cox, J.; Mann, M.; Horning, S., Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer. Mol Cell Proteomics 2011, 10 (9), M111 011015. Gallien, S.; Duriez, E.; Crone, C.; Kellmann, M.; Moehring, T.; Domon, B., Targeted proteomic quantification on quadrupole-orbitrap mass spectrometer. Mol Cell Proteomics 2012, 11 (12), 1709-23. Mani, D. R.; Abbatiello, S. E.; Carr, S. A., Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics. BMC bioinformatics 2012, 13 Suppl 16, S9.

492 493

Figure 2. Linear range of the 8-plex MS-based immunoassay for one porcine and one bovine meat and

494

bone meals (MBM) spiked in fish feed (FF) after separate digestion (n=3).

20


Page 21 of 26


TABLES

Alpha-2-Macroglobulin

Factor VIII

Antithrombin III

Serum Albumin

Cholinesterase

9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9 8/9

anas platyr.

anser anser

meleagris gallopavo

gallus gallus

X

equus caballus

X

sus scrofa

capra hircus

GSGGTAEHPFTVEEFVLPK ESGGTAEHHFTVEEFVLPK VVVQQESGETAEHPFTVEEFVLPK AEHPFIVEEFVLPK TIHHPFSVEEYVLPK TIQHPFTVEEYVLPK TIQHPFSVEEYVLPK IQHSFSVEEYVLPK SWAHHIALR IHPQSWVHHIALR SWVHHIALR IHPTSWAQHIALR IHPQSWGHQIALR HWHNHIALR IHPAWHNHIALR QWHNHIALR ITDVIPPQAINEFTVLVLVNTIYFK ITDVIPPQAIDEFTVLVLVNTIYFK ITDVIPPEAINELTVLVLVNTIYFK ITDVIPHGAINELTVLVLVNTIYFK GIDDLTVLVLVNTIYFK GIDELTVLVLVNTIYFK ITEVIPEGGINDLTVLVLVNTIYFK DAFLGSFLYEYSR DVFLGSFLYEYSR HVFLGTFLYEYSR DVFLGTFLYEYSR SFEAGHDAFMAEFVYEYSR SFEAGHDAFMSEFVYEYSR FSDMGNNAFFYYFEHR FSEMGNNAFFYYFEHR FSELGNDAFFYYFEHR TIAEVGNNVFFYFFEHR IAEIGNNVFFYFFEHR FAQLGHNAFFYFFEHR

ovis aries

peptide sequence

bos taurus

Protein

species coverage

Table 1. Target proteins and their homologous peptides in different species identified by bioinformatics.

X X X X X X X X X X X X X X X X X

X X X X X X

X X

X X X X X

X

21


X

X

X X X X X X


Page 22 of 26

Table 2. Linearity and sensitivity of the 8-plex MS-based immunoassay determined by a dilution series of standard peptides in digested fish feed as matrix (n=3). Species Cattle Sheep/Goat Pig Horse Chicken Turkey Duck Goose

Slope 0.46 0.74 1.14 1.10 0.86 0.75 0.74 0.62

PRM Intercept -5.94E-03 -1.02E-02 +8.90E-03 -7.45E-03 -1.51E-02 -1.35E-02 -1.18E-02 -2.13E-02

R² 0.9989 0.9962 0.9927 0.9908 0.9993 0.9999 0.9984 0.9997

LOD / amol 30 245 692 167 155 235 116 431

Slope 0.99 0.71 0.96 1.08 0.80 0.77 1.06 0.85

SIM Intercept -0.32E-01 +3.92E-05 +3.22E-03 +2.99E-03 +1.47E-03 +5.93E-03 -5.38E-02 1.11E-02

R² 0.9999 0.9866 0.9997 0.9998 0.9996 0.9997 0.9972 0.9998

LOD / amol 486 336 1200 1025 340 657 248 11862

Table 3. Leave-one-out assay specificity determined by the analysis of native plasma mixtures (10%, w/w.) in digested fish feed as matrix (n=3). Sample / Species No Pig No Cattle No Sheep/Goat No Horse No Turkey No Goose No Duck No Chicken All Species in FF All Species in PBS FF, no Plasma PBS, no Plasma

Pig y20++

Cattle y17++ 8 462 426 405 401 409 436 402 421 412 8 1

241 0 255 258 231 259 255 261 246 238 0 0

Mean peptide amount in nmol/g Sheep/Goat Horse Turkey Goose y17++ y7+ y7+ y7+ 130 248 84 66 129 265 85 67 0 268 82 73 140 0 84 72 158 277 0 67 158 276 81 0 167 276 87 70 160 276 85 71 145 266 86 67 146 263 86 70 0 0 0 0 0 0 0 0

22


Duck y7+ 56 61 59 64 62 62 0 63 63 60 0 0

Chicken y7+ 119 112 119 112 119 116 114 0 112 104 0 0

Page 23 of 26


Table 4. Assay specificity determined by the analysis of animal protein samples of different species and tissue origin (n=3).

Sample

Cattle y17++

FF (no mammals) FF (with mammals) Poultry BM Poultry MBM MBM Cattle Porcine Meal 1 Porcine Meal 2 Porcine Meal 3 Porcine Meal 4 Porcine BM Porcine SDP 1 Porcine SDP 2 Bovine SDP Unknown SDP

Sheep y17++

0 0 0 0 2.5 0 0 0 0 0 0 0 185.0 151.2

0 0 0 0 0 0 0 0 0 0 0 0 0 0

Mean peptide amount in nmol/g Pig Horse Turkey Chicken y20++ y7+ y7+ y7+ 0.04 0 0 0 25.8 0 0 0.5 0 0 2.9 11.4 0 0 0 0.5 0 0 0 0 6.1 0 0 0 5.3 0 0 0 6.9 0 0 0 9.2 0 0 0 113.1 0 0 0 159.6 0 0 0 294.4 0 0 0 0 0 0 0 37.1 0 0 0

Duck y7+

Goose y7+ 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

Table 5. Intra- and inter-assay precision of the 8-plex MS-based immunoassays determined by the measurement of a plasma mix (9 species) in fish feed at three concentrations (1%, 5% and 10%, w/w), prepared on the dried and non-digested level (n=5).

Species Cattle Sheep/Goat Pig Horse Chicken Turkey Duck Goose

10% w/w (1.1% per species) mean / CV fmol /% 871 6 1423 10 1104 5 500 8 189 7 113 8 80 4 129 5

Intra assay precision 5% w/w (0.6% per species) mean / CV fmol /% 459 5 651 7 571 6 248 8 103 5 54 5 40 8 68 3


Inter assay precision 10% w/w 5% w/w (1.1% per (0.6% per species) species) mean / CV mean / CV fmol /% fmol /% 892 4 461 7 1351 12 658 9 1110 6 588 3 526 5 300 7 203 6 103 16 121 4 66 14 85 9 48 8 132 4 68 17

23




FIGURE GRAPHICS Figure 1:

24


Page 24 of 26

Page 25 of 26


Figure 2:

482

25



483

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GRAPHIC FOR TABLE OF CONTENTS

484 485

CORRESPONDING AUTHOR

486

Dr. Oliver Poetz, SIGNATOPE GmbH, 72770 Reutlingen

487

E-mail [email protected]; phone +49 (0)7121 744086-1.

488 489

AUTHOR CONTRIBUTIONS

490

A.E.S. was in charge of the experimental work. The manuscript was written through contributions of

491

all authors. All authors have given approval to the final version of the manuscript.

492 493

CONFLICT OF INTEREST

494

HP, TOJ and OP are shareholders of SIGNATOPE GmbH. SIGNATOPE offers assay development and

495

service using MS-based immunoassay technology.

496

26


Species Differentiation and Quantification of Processed Animal

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