Development and Validation of a Multiplexed Protein Quantitation

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Journal of Agricultural and Food Chemistry

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1

Development and Validation of a Multiplexed Protein Quantitation Assay

2

for the Determination of Three Recombinant Proteins in Soybean Tissues by

3

Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS)

4

Ryan C. Hill†, Trent J. Oman†, Guomin Shan†, Barry Schafer†, Julie Eble*, and Cynthia Chen*

5

†

6

*

7

United States.

Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States

Critical Path Services LLC, 3070 McCann Farm Drive, Garnet Valley, Pennsylvania 19060,

ACS Paragon Plus Environment


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Abstract:

9

Currently,

traditional

immunochemistry

technologies

such

as

Enzyme-Linked

10

Immunosorbent Assays (ELISA) are the predominant analytical tool used to measure

11

levels of recombinant proteins expressed in genetically engineered (GE) plants. Recent

12

advances in agricultural biotechnology have created a need to develop methods capable

13

of selectively detecting and quantifying multiple proteins in complex matrices due to

14

increasing numbers of transgenic proteins being co-expressed or “stacked” to achieve

15

tolerance to multiple herbicides or to provide multiple modes of action for insect control.

16

A multiplexing analytical method utilizing liquid chromatography with tandem mass

17

spectrometry (LC-MS/MS) has been developed and validated to quantify three herbicide

18

tolerant proteins in soybean tissues; aryloxyalkanoate dioxygenase (AAD-12), 5-enol-

19

pyruvylshikimate-3-phosphate

20

acetyltransferase (PAT).

21

precision over multiple analysts and laboratories. Results from this method were

22

comparable to those obtained with ELISA with respect to protein quantitation, and the

23

described method was demonstrated to be suitable for multiplex quantitation of

24

transgenic proteins in GE crops.

25

KEYWORDS: Multiplex, LC-MS/MS, ELISA, Surrogate Peptide, Biotechnology, GE

26

Crops, Protein Quantitation, Stacked Trait Product, Validation, Digestion Efficiency,

27

Recombinant Protein.

synthase

(2mEPSPS),

and

phosphinothricin

Results from the validation observed high recovery and


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Introduction:

29

In an effort to feed an estimated population of 9 billion by 2050, a 70 % rise in food

30

consumption1 is expected, along with an increasing demand for biofuels2. Advancements

31

in agricultural biotechnology have introduced increasing numbers of genetically

32

engineered (GE) crops globally to combat plant pests, weeds, and diseases that reduce

33

crop yields.

34

documented to reduce pesticide use3, 4. As such, GE crops can be one tool enabling

35

future agricultural needs to be met.

36

The desire to have single crop varieties with multiple beneficial GE traits is resulting in

37

increasing numbers of transgenic proteins being co-expressed or “stacked” together to

38

achieve tolerance to multiple herbicides5 or to provide multiple modes of action to delay

39

insect resistance6. These stacked trait products act to prevent or delay the emergence or

40

development of resistance to the individual toxins or herbicides and improve pest

41

mangement7, 8. Accurate quantitation of these transgenic proteins from complex matrices

42

is needed to support product development, risk assessment, registration, breeding, and

43

production9,

44

antibody based immunochemistry technologies such as Enzyme-Linked Immunosorbent

45

Assays (ELISA) to detect and quantify proteins introduced into different varieties of

46

plants and crops11. Common attributes of an ELISA include high sensitivity, specificity,

47

and once the method has been developed are relatively inexpensive for routine analysis.

48

However, ELISA method development is often time consuming and challenging

49

(exhaustive protein extraction with ELISA compatible buffers, cross-reactivity due to

50

sequence homology of endogenous proteins and target proteins, etc.) in addition to being

When managed correctly, the selectivity of these products has been

10

.

To date, the Ag-Biotech industry has relied heavily on the use of



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most suitable for use in a single protein analysis format. The trend in industry toward

52

plant varieties expressing multiple transgenic proteins makes immuno-detection very

53

challenging due to the increasing volume of data needed to be acquired per sample. As an

54

example SmartStax® corn, which expresses 8 unique proteins12, requires 8 individually

55

developed ELISA methods to analyze each sample.

56

Although immunochemistry methods for protein detection have long been accepted in the

57

Ag-Biotech industry13,

58

requires consideration of alternative analytical methods for protein detection and

59

quantitation.

60

MS/MS) has been common practice for detection and quantitation of small molecules in

61

pharmaceuticals15,

62

signature peptide analysis by LC-MS/MS as surrogates for target intact proteins has been

63

proposed in previous literature22, 23. To demonstrate signature peptide quantitation by

64

LC-MS/MS as a suitable multiplex platform to quantify multiple proteins within a single

65

analysis, we report a comprehensive development and validation of a multiplex LC-

66

MS/MS method for the determination of the aryloxyalkanoate dioxygenase (AAD-12), 5-

67

enol-pyruvylshikimate-3-phosphate

68

acetyltransferase (PAT) herbicide tolerant proteins in Enlist E3™ soybean tissues.

69

Materials and Methods:

70

Materials: Soybean plants were grown in a Dow AgroSciences greenhouse (Indianapolis,

71

IN), and leaf, root, and seed tissues were lyophilized, ground to a fine powder, then

72

stored at -80 °C. Recombinant protein standards were expressed in and purified from

73

Pseudomonas fluorescens (>95% purity) and aliquots were stored at -80 °C for single

14

; the increasing complexity of stacked transgenic products

Liquid chromatography coupled to tandem mass spectrometry (LC-

16

as well as pesticide17-19 and herbicide residues20, 21. The use of

synthase

(2mEPSPS),

and

phosphinothricin

SmartStax® is a trademark of Monsanto Technology LLC. SmartStax® multi-event technology developed by Dow AgroSciences and Monsanto Enlist E3™ is a registered trademark of The Dow Chemical Company (“Dow”) or an affiliated company of Dow. Enlist E3™ soybeans are jointly developed by Dow AgroSciences and M.S. Technologies, L.L.C.


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usage with protein concentrations assessed by amino acid analysis using acid hydrolysis,

75

automated OPA/FMOC derivatization, and C18 reverse-phase separation with

76

fluorescence detection. Synthetic peptides representative of natural abundance peptides

77

from each protein, defined as surrogate peptides, and heavy labeled peptide internal

78

standards (L-Lysine-13C6,

79

were obtained from New England Peptide (Gardner, MA). Phosphate buffered saline

80

containing 0.05% Tween 20 (PBST) was obtained from Teknova (Hollister, CA). Casein

81

was purchased from SurModics (Eden Prairie, MN). Albumin chicken egg (OVA),

82

polyvinylpyrrolidone (PVP), and HPLC solvents were obtained from Sigma Aldrich (St.

83

Louis, MO). Ammonium bicarbonate, dithiothreitol, and formic acid were obtained from

84

Fisher Scientific (United States). Sequencing grade trypsin was purchased frozen from

85

Promega Corporation (Madison, WI).

86

LC-MS/MS Method Overview: All surrogate peptide LC-MS/MS method development

87

and validation was performed at Dow AgroSciences (Indianapolis, IN). Briefly, a total of

88

1.5 mL of PBST and 2 metal beads were added per 15 mg of lyophilized soybean leaf,

89

root, or seed tissue weighed into low binding microcentrifuge tubes. Samples were

90

extracted in a bead grinder (Geno/Grinder, Swedesboro, NJ) at 1500 strokes per minute

91

for 3 minutes followed by centrifugation at 4 °C and > 3000 rpm for 10 minutes. A total

92

of 100 µL of supernatant was transferred to a clean microcentrifuge tube with the

93

addition of 7.5 mM dithiothreitol (DTT) and incubated at 95 °C for 45 minutes followed

94

by cooling at 4 °C. After the samples were cooled to room temperature, 60 µL 50 mM

95

ammonium bicarbonate, 20 µL heavy isotope labeled peptide internal standard, and 5 µg

96

trypsin enzyme (enzyme/substrate ratio ~1:75 (w/w) based on BCA assay results for total

15

N2 or L-Arginine-13C6,

15

N4 depending on protein sequence)



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extractable protein content from respective tissues) were added. The synthetic natural

98

abundance peptide reference standards were spiked into control soybean tissue and

99

processed the same as the unknown samples.

The samples and peptide reference

100

standards were incubated overnight at 37 °C followed by quenching with formic

101

acid/H2O (50/50, v/v). The samples were centrifuged at > 3000 rpm for 10 minutes to

102

pellet any undigested insoluble particulate followed by LC-MS/MS analysis.

103

Quantitation of detected surrogate peptides was performed with linear regression and 1/x2

104

weighting followed by conversion into respective protein concentrations using the

105

following

106

Protein Concentration = Detected Peptide Concentration x ቀPeptide Molecular Weightቁ.

107

The LC-MS/MS system included an AB Sciex 4000 QTRAP with turbo ion-spray source

108

and Waters Acquity H-Class UPLC. The autosampler temperature was kept at 4 °C

109

during analysis. A total of 5 µL of sample was injected onto an Acquity UPLC BEH130

110

C18 1.7 µm (2.1 x 50 mm) column set at 50 °C. The reverse phase analysis was

111

performed using an organic mobile phase (MPB) acetonitrile containing 0.1 % formic

112

acid and the aqueous mobile phase (MPA) was water containing 0.1 % formic acid. The

113

LC flow rate was 0.5 mL/min with a linear gradient from 0.1 % MPB to 28 % MPB over

114

6 minutes, followed by 100 % MPB for 1 minute to wash the column, and 0.1 % MPB for

115

1 minute to re-equilibrate the column. The needle wash and seal wash contained 50/50

116

methanol/water with no detected carryover between samples.

117

The mass spectrometer was operated in positive ion mode with ionspray voltage of 5500

118

volts and temperature set at 450 °C. MS/MS transitions and collision energy for each

equation

and

information

found

in

Table

1:

Protein Molecular Weight


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peptide can be found in Table 1. Other relevant instrument parameters included curtain

120

gas of 35 psi, ion source gas 1 and 2 of 55 psi, collision gas set to medium, entrance

121

potential of 10 volts, declustering potential of 80 volts, and collision cell exit potential of

122

15 volts.

123

LC-MS/MS Method Validation:

124

Extraction Efficiency (EE): EE was evaluated by repeatedly extracting transgenic

125

soybean tissue samples containing AAD-12, 2mEPSPS, and PAT proteins and

126

determining the amount of protein from each extraction. After extraction, the samples

127

were centrifuged and the supernatant was transferred to a new vial and the extraction of

128

the pellet was repeated. Each extract was analyzed for surrogate peptide concentrations

129

by LC-MS/MS and the levels of transgenic proteins in the extracts and the pellet were

130

confirmed by western blotting. The extraction efficiency for each protein in each tissue

131

was calculated as the percentage of peptide detected in the first extraction relative to the

132

peptide found in all soluble extractions by the following equation:

133

EE = ቀSum of peptide recovered from all extractionsቁ x 100).

134

Western Blotting: Briefly, 750 µL Laemmli buffer was added to each pelleted tissue after

135

the 5th serial extraction and extracted with the Geno Grinder at 1500 strokes/min for 3

136

minutes followed by centrifugation at 4 °C/>3000 rpm for 5 minutes. The first and 5th

137

extraction supernatants were diluted 1:2 into 2× concentrated Laemmli buffer, all

138

prepared solutions were heated at 95 °C for 10 minutes, loaded onto a gel along with

139

recombinant protein standards with XT MES running buffer, and separated by

140

electrophoresis for approximately 1 hour at 160V. Each gel was transferred to a

Amount of peptide recovered in 1st extraction



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nitrocellulose membrane, washed 3 times with PBST, and incubated with each

142

corresponding primary antibody in PBST overnight at 4 °C with gentle shaking. After

143

primary antibody incubation, the membranes were washed 3 times with PBST. The

144

membranes were then incubated with respective HRP-conjugated secondary antibodies

145

prepared in blocking solution for 1 hour at room temperature with gentle shaking. After

146

incubation with secondary antibodies, the membranes were washed 3 times with PBST.

147

The membranes were exposed to X-ray film after addition of chemilumenscent substrate

148

to the membrane.

149

Accuracy: Method accuracy was assessed by determining the recovery of surrogate

150

peptides (described in Table 1) produced from the digestion of AAD-12, 2mEPSPS, and

151

PAT recombinant proteins that had been fortified into non-GE control soybean tissues at

152

the proposed limit of quantitation, a mid-range concentration, and a high-range

153

concentration for each protein. Briefly, control soybean tissue (leaf, root, or seed

154

depending on which matrix was being evaluated) was extracted with PBST and the

155

supernatant was pooled together. Each pooled tissue extract was then fortified with all

156

three proteins, denatured, digested, and analyzed by LC-MS/MS. Recovery was

157

determined as a percentage of fortified recombinant proteins by Detected Peptide Concentration x ቀ

Protein Molecular Weight ቁ Peptide Molecular Weight

158

% Recovery =

159

Precision and Ruggedness: Precision and ruggedness were determined by evaluating the

160

recovery of surrogate peptides produced from the digestion of AAD-12, 2mEPSPS, and

161

PAT recombinant proteins that had been fortified into control soybean tissues at the limit

Theoretical Protein Fortification Concentration

× 100.


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of quantitation, mid-range and high-range concentration for each protein across multiple

163

days.

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Peptide Stability: Digested peptide stability was examined over the course of 5 days. A

165

sample set of non-GE control tissues fortified with AAD-12, 2mEPSPS, and PAT

166

recombinant proteins at the limit of quantitation, mid-range, and high-range concentration

167

was digested, and analyzed along with reference peptide standards prepared in control

168

tissue by LC-MS/MS (Day 0). The reference standard peptides and fortified tissue

169

samples were stored at 4 °C for 5 days and reanalyzed using the same instrument

170

parameters as the day 0 analysis. The difference in recovery of peptides from day 0 to

171

day 5 was assessed.

172

Sensitivity: The preliminary quantitative range for the LC-MS/MS multiplex assay was

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empirically defined on the basis of assay parameters such as analyte signal intensity,

174

background noise intensity, matrix contributions, and analyte stability from an overnight

175

digest. Three times the signal to noise ratio and ten times the signal to noise ratio of

176

peptide standard response to digested control sample were used to define LOD and LOQ

177

respectively (Table 2). The calibration standard range was defined to be at least 20%

178

above and below the quantitative range for all proteins with acceptable correlation

179

coefficients ≥ 0.995.

180

Interlaboratory Method Assessment:

181

method was performed at Critical Path Services, LLC (Garnet Valley, PA). Control

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soybean tissues described in the materials section were fortified with recombinant AAD-

183

12, 2mEPSPS, and PAT proteins at the limit of quantitation, mid-range and high-range

An independent assessment of the LC-MS/MS



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concentrations and analyzed following the method described in “LC-MS/MS Method

185

Overview”.

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coupled to an Agilent 6410B mass spectrometer.

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ELISA and LC-MS/MS Comparison: Leaf tissue containing AAD-12, 2mEPSPS, and

188

PAT proteins from nine individual soybean plants was analyzed using ELISA and LC-

189

MS/MS. The leaf samples were analyzed by LC-MS/MS following the analytical method

190

described in “LC-MS/MS Method Overview”.

191

methods were developed and validated under Good Laboratory Practice guidelines for

192

each protein. For each ELISA method described below, a total of 1.5 mL of respective

193

extraction buffer and 2 metal beads were added to 15 mg of soybean leaf tissue and

194

extracted in a Geno/Grinder at 1500 strokes per minute for 3 minutes and then

195

centrifuged at 4 °C/>3000 rpm for 5 minutes. The assays were measured using purified

196

recombinant protein as standards with quadratic regression.

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The AAD-12 protein was extracted from soybean leaf tissue with phosphate buffered

198

saline solution containing 0.05% Tween 20 (PBST) buffer with 0.75% ovalbumin. The

199

extract was centrifuged; the aqueous supernatant was collected, diluted, and assayed

200

using a specific AAD-12 ELISA kit (Envirologix, Portland Maine). An aliquot of the

201

diluted sample was incubated with enzyme-conjugated anti-AAD-12 protein monoclonal

202

antibody in the wells of an anti-AAD-12 polyclonal antibody coated plate. At the end of

203

the incubation period, the unbound reagents were removed from the plate by washing

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with PBST followed by incubation of enzyme conjugate with an enzyme substrate. The

205

reaction was quenched with acid and the absorbance was read at 450 nm minus

206

absorbance at 650 nm using a plate reader.

The LC-MS/MS analysis was performed on an Agilent 1290 UHPLC

Individual sandwich based ELISA


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The 2mEPSPS protein was extracted from soybean leaf tissue with a phosphate buffered

208

saline solution containing 0.05% Tween 20 and 2× Casein. The extract was centrifuged;

209

the aqueous supernatant was collected, diluted with PBST/Casein, and assayed using a

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specific 2mEPSPS ELISA kit made in house at Dow AgroSciences. An aliquot of the

211

diluted sample was incubated in the wells of an anti-2mEPSPS polyclonal antibody

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coated plate, and then the unbound samples were removed from the plate by washing

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with PBST. An excess amount of enzyme-conjugated anti-2mEPSPS protein monoclonal

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antibody was subsequently added to the wells. At the end of the incubation period, the

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unbound reagents were removed from the plate by washing with PBST. Subsequent

216

addition of an enzyme substrate generated a colored product. The reaction was quenched

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with acid and the absorbance was read at 450 nm minus absorbance at 650 nm using a

218

plate reader.

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The PAT protein was extracted from soybean leaf tissue with a phosphate buffered saline

220

solution containing 0.05% Tween 20 and 1% polyvinylpyrrolidone (PBST/PVP). The

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extract was centrifuged; the aqueous supernatant was collected, diluted and assayed using

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a specific PAT ELISA kit (Envirologix, Portland Maine). An aliquot of the diluted

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sample was incubated with enzyme-conjugated anti-PAT protein monoclonal antibody in

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the wells of an anti-PAT polyclonal antibody coated plate in a sandwich ELISA format.

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At the end of the incubation period, the unbound reagents are removed from the plate by

226

washing with PBST. The presence of PAT was detected by incubating the antibody-

227

bound enzyme conjugate with an enzyme substrate, generating a colored product. The

228

reaction was quenched with acid and the absorbance was read at 450 nm minus

229

absorbance at 650 nm using a plate reader.



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Results and Discussion

231

Method Development:

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Target Peptide Selection:

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known cleavage specificity and a protein’s primary sequence to cleave high molecular

234

weight proteins into smaller peptide chains of predicted sequence suitable for analysis by

235

tandem mass spectrometry24.

236

representative of the target proteins25. The enzyme trypsin was chosen for this assay due

237

to its high specificity to cleave at the C-terminus of lysine and arginine residues. For

238

method development, all possible tryptic peptides for each target protein from a

239

theoretical digestion were considered and surveyed using specific criteria during the

240

peptide selection process. Initial selection criteria required 6-20 amino acids in length, as

241

fewer than 4 residues can lead to nonspecific identification26.

242

susceptible to modification such as those produced from N-terminal cleavage or residues

243

prone to oxidation such as methionine and cysteine were excluded from selection.

244

Digested recombinant protein standards were analyzed using a targeted information-

245

dependent acquisition (IDA) in the mass spectrometer to determine relative fragmentation

246

ratios and highest abundance MS/MS fragment ions for each tryptic peptide candidate.

247

Using the most abundant MS/MS fragmentation ions and considering charge state,

248

peptides were further profiled for sensitivity and reproducibility from an overnight

249

digestion, and minimal isobaric interference in comparison to control tissue (specificity).

250

Two surrogate peptides were chosen per protein for further optimization representing

251

different locations of the protein sequence to prevent missed identification during

Traditional bottom-up proteomics employs a protease of

These peptides are defined as surrogate peptides


Peptide residues

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analysis due to truncation. One peptide was defined as the quantitation peptide with the

253

second peptide serving as a confirmation peptide during quantitation (Table 1).

254

Extraction: The LC-MS/MS extraction mirrored individual ELISA methods for AAD-

255

12, 2mEPSPS, and PAT to incorporate direct comparison of the assays. Phosphate

256

buffered saline with 0.05 % Tween 20 is the base component of each of the ELISA

257

methods. Tissue containing AAD-12, 2mEPSPS, and PAT proteins was extracted with

258

different extraction buffers offering a large range of biochemical properties (PBST, 50

259

mM Tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0), or 50 mM ammonium

260

bicarbonate), digested, and analyzed by LC-MS/MS. Results demonstrated little change

261

in peptide response confirming PBST as suitable for extraction for all three proteins in

262

the multiplex method (Figure 1). Many ELISA extraction buffer components (non-

263

volatile detergents such as Tween 20, surfactants, etc.) are less favorable for extended

264

LC-MS/MS analysis27. As surrogate peptide analysis decouples from accepted ELISA

265

methodology, further optimization of extraction buffers to reduce surfactants and

266

detergents or removal of these components all together before analysis can improve

267

sensitivity for detection by LC-MS/MS. The optimized amount of tissue per volume

268

buffer and buffer choice for the multiplex LC-MS/MS method was 15 mg lyophilized

269

tissue per 1.5 mL PBST.

270

Digestion: The underlying foundation for surrogate peptide analysis relies on protease

271

digestion that is 100% efficient in order for the resulting peptide fragments to be present

272

in equal molar amounts compared to the original target protein prior to digestion.

273

Proteins can differ greatly in their susceptibility to proteolysis, and this requires a

274

digestion protocol that is optimal for all proteins in a multiplex analysis for accurate



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measurements. Prior to digestion, proteins were denatured to primary structure at high

276

temperatures and reduced in the presence of DTT to allow for efficient proteolytic

277

cleavage.

278

temperature and time incurred lower accuracy from recombinant protein spike recovery.

279

The trypsin concentration, peptide stability over a particular digest time interval, and

280

buffer selection (pH) were key variables in the digestion development.

281

enzyme-substrate ratio of 1:75 enabled 100% digestion efficiency, as increasing the

282

concentration of trypsin did not increase detected peptide response from the overnight

283

digestion.

284

enzymatic digestion efficiency and downstream LC-MS/MS analysis28. To minimize

285

these effects and achieve an optimal digestion condition for trypsin (pH 7.5 - 8.5), the

286

denatured supernatant was diluted into 50 mM ammonium bicarbonate prior to addition

287

of trypsin enzyme for the overnight digestion.

288

A time-course evaluation for digestion efficiency has been documented in literature to

289

demonstrate digestion completeness29; the abundance of target signal and stability of

290

signal over the length of time of the digest were considered. Individual replicates were

291

extracted in PBST, digested, and quenched with acid at 3 time points (4, 10, and 15 hour

292

to simulate an overnight digestion) and peak area ratios were plotted versus digestion

293

time (Figure 2).

294

digestion time did not improve detection and the signal was stable at time points

295

representative of an overnight digestion. An enzyme-substrate ratio of 1:75 and 12 hour

296

digestion time were selected for the method.

Optimized conditions were found to be 95 °C for 45 minutes as lower

A trypsin

Detergents, such as Tween 20 used in the extraction buffer, can impact

Confidence in digestion completeness can be inferred as longer


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Reference Standards:

Representative synthetic peptides were chosen as reference

298

standards instead of recombinant proteins which are commonly used in ELISA analysis.

299

Digested recombinant protein standards may provide a linear quantitative range, however

300

due to reliance on proteolysis some intrinsic variability will exist from analysis to

301

analysis adding complexity to interpretation of results. Once completeness of digestion

302

and peptide stability have been validated for a particular trypsin concentration and

303

digestion time interval, the use of synthetic peptide standards may offer a more straight

304

forward approach to quantifying incurred peptide residues from proteolysis. Initially the

305

reference peptide standards were prepared in extraction buffer, however peptide loss was

306

observed due to surface absorption of more hydrophobic peptides to labware during the

307

sample preparation affecting the accuracy of the standard curve (Figure 3)30.

308

overcome these effects, reference standards were prepared in digested control soybean

309

matrix in which native proteins provide a protective medium to minimize peptide surface

310

absorption. Proteins such as bovine serum albumin or ovalbumin are commonly used as

311

buffer additives in the same intent as the digested control matrix.

312

characterization of materials used during sample treatment may allow for the standard

313

curve to be prepared in extraction buffer alone simplifying the analytical procedure.

314

Multiplexing Assay: The multiplex detection of AAD-12, 2mEPSPS, and PAT surrogate

315

peptides in reference standards and unknown samples was performed by tandem mass

316

spectrometry. The term SRM or selected reaction monitoring refers to the selectivity of

317

tandem mass spectrometry where a precursor ion (in this case peptide) with specific mass

318

to charge ratio (m/z) is identified and fragmented into one or more product ions or

319

MS/MS transitions.

To

Further

SRM LC-MS/MS based detection is specific by virtue of



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chromatographic separation (Figure 4) and selective MS/MS detection. A second layer

321

of specificity is produced from monitoring multiple product ions per peptide defined as a

322

confirmation ratio31. The confirmation ratio represents the relative abundance between

323

two product ions derived from the same precursor ion demonstrating real-time validation

324

of results.

325

structurally identical heavy labeled peptide internal standard (HIS) to normalize all

326

samples and reference standards and reduce the impact of common technical pitfalls such

327

as signal suppression or enhancement (matrix effects), ion drift, among others. The

328

precursor and product ions of the HIS should co-elute with those of the natural abundance

329

peptide producing a peak area ratio32. The peak area ratio in the unknown sample is

330

interpolated to the peak area ratios produced from the reference standard curve for a

331

quantitative result.

332

standards normalizes results across different analytical runs and instruments as the

333

quantitation is not strictly limited to the intensity of signal leading to more reproducible

334

data and a greater degree in confidence for interpretation of results. Although only one

335

peptide is typically reported for quantitation results, two peptides are monitored per

336

protein in the analytical method to confirm complete digestion during sample analysis.

337

Method Validation Results:

338

Extraction Efficiency (EE): The extraction was found to be effective with efficiencies for

339

all proteins between 90-100% for all tissues (Figure 5a). Western blotting was used to

340

qualitatively confirm the extraction results and demonstrated that the surrogate peptides

341

were truly representative of intact AAD-12, 2mEPSPS, and PAT proteins in plant

342

extracts (Figure 5b). No immunoreactivity was observed in the last serial extraction

Further specificity from the method is obtained from the addition of a

The use of peak area ratios of unknown samples to reference


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supernatant or in the pelleted tissue after the last extraction as evidence supporting

344

effective extraction.

345

Accuracy:

346

fortifications reinforces complete digestion and supports the use of synthetic peptide

347

reference standards as a calibration source.

348

percentage peptide recovery from recombinant protein fortifications performed across

349

multiple days.

350

2mEPSPS, and PAT with observed mean recoveries for all tissues between 70-123%

351

(Table 3).

352

Precision and Ruggedness: Precision of the multiplex method was evaluated across two

353

separate analyses for each tissue. To avoid any potential loss during extraction impacting

354

precision results, control samples were extracted with buffer, pooled together, and

355

fortified with recombinant protein standards. The precision results showed in general

356

CVs ≤ 20% for both inter- and intra- day analysis for all three proteins (Table 3).

357

Specificity/Matrix Effects:

358

differentiate a target analyte from other components in a complex matrix. Evaluations for

359

ELISA commonly address assay specificity by spiking non-target proteins into control

360

tissue at a range of concentrations to determine the level of immunoreactive cross-

361

reactivity and through comparison of standard curves prepared in different dilutions of

362

extracted control tissue to standard curves prepared in buffer to assess matrix effects.

363

Surrogate peptide LC-MS/MS specificity should be addressed during peptide selection in

364

method development with evaluation of recombinant protein standards fortified into

Good recovery of surrogate peptides from intact recombinant protein

The assay accuracy was indicated as

The accuracy of the multiplex method is acceptable for AAD-12,

Specificity is the ability of an analytical method to



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control tissue, unfortified control tissues, and tissues containing the protein of interest

366

(Figure 6). Following peptide selection, assay specificity is not as great of an issue with

367

LC-MS/MS by virtue of heavy labeled peptide internal standards to normalize all samples

368

(not as the quantitation source) and the aforementioned chromatographic separation

369

coupled to MS/MS detection.

370

Peptide Stability: In addition to peptide stability from an overnight digestion (evaluated

371

from the time-course digestion and fortification experiments), digested peptide stability

372

in storage conditions over a length of time was assessed to incorporate sample

373

reinjection, instrument issues, etc.

374

recombinant proteins in control tissue were confirmed to be stable for at least 5 days

375

when stored at 4 °C with acceptable mean recoveries of 70-120% (Table 4).

376

Sensitivity: The limit of detection is defined as the analyte concentration that gives a

377

significant difference from the analyte-free sample. The quantitative range is generally

378

defined as the highest and lowest concentrations which can be determined with an

379

acceptable degree of accuracy. The quantitative range was proposed during method

380

development to encompass the general protein levels observed in transgenic plants rather

381

than reflecting the true assay quantitative range.

382

instrumentation has been shown to span 3-5 orders of magnitude33 which can be further

383

optimized to increase the defined upper range of quantitation for the assay. The targeted

384

LOD and LOQ (Table 2) for each protein were empirically defined based on assay

385

parameters such as peptide signal intensity and signal-to-noise ratio. In addition to

386

accuracy of recovery, the LOD and LOQ were also verified by statistical approaches34

387

using 3× and 10× the standard deviation respectively from the LOQ recovery results for

The digested AAD-12, 2mEPSPS, and PAT

The dynamic range of LC-MS


Page 19 of 43


Page 19 388

each protein (Table 3). The statistically calculated LOD and LOQ results were less than

389

the target LOD and LOQ results for AAD-12 and PAT in all tissues as well as leaf tissue

390

for 2mEPSPS (Table 3). Statistical calculations of 2mEPSPS were slightly higher for

391

root and seed due to low recovery in 1 sample from each analysis potentially due to

392

previously mentioned peptide absorption to materials during sample preparation.

393

Overall, the results provide appropriate estimates of the quantitative range of the assay.

394

Interlaboratory Validation: Independent laboratory assessment of accuracy and precision

395

of the LC-MS/MS method was performed with AAD-12, 2mEPSPS and PAT

396

recombinant proteins fortified into non-GE control tissues by Critical Path Services LLC.

397

Recovery results were comparable to data obtained from the full validation performed by

398

Dow AgroSciences (Tables 3 and 5) and demonstrated LC-MS/MS as a robust workflow

399

incorporating different analysts and instrumentation (vendor, ionization source, and

400

detector) with reproducible results. In addition, minimal optimization was performed to

401

the original MS/MS instrument parameters during the independent laboratory assessment

402

yielding similar results between laboratories, demonstrating the ease of transferring the

403

methodology (as has been previously documented in literature35).

404

ELISA and LC-MS/MS Comparison: ELISA immunochemistry uses antibodies highly

405

specific to target proteins.

406

commonly from the addition of an enzyme substrate to a specific antibody “sandwich”

407

containing target protein producing a colored product. The resulting colored product,

408

measured as optical density, is proportional to the concentration of protein in the sample

409

which is correlated to the optical densities produced from reference standards.

410

contrast, SRM LC-MS/MS based detection is specific by virtue of the chromatographic

Quantitation depends on a single signal produced most


In


Page 20 of 43

Page 20 411

separation and selective MS/MS detection. SRM LC-MS/MS employs multiple signals

412

to evaluate the concentration of target analyte such as MS/MS confirmation ratios, peak

413

area ratios, and monitoring multiple peptides representing multiple areas of the protein

414

sequence providing greater confidence in obtained results36. The samples and natural

415

abundance peptide reference standards are standardized by virtue of heavy labeled

416

peptide internal standards increasing the precision and accuracy across multiple analysis

417

compared to ELISA. In addition to multiplex capability, the increased dynamic range of

418

LC-MS/MS compared to ELISA (generally 3-5 orders of magnitude compared to 2-3)

419

allows for fewer reanalysis of samples due to the need for sample dilution into the

420

reference standard range. The LOQ for LC-MS/MS was comparable to the individual

421

ELISA assays (Table 2).

422

ELISA measures immunoreactivity of intact proteins largely depending on protein

423

conformation contrary to surrogate peptide LC-MS/MS which measures total abundance

424

of denatured linear peptide chains where unstable protein in tissues will have less impact

425

on quantitation results. Due to the complexity and intrinsic variability of biological

426

analysis which is present in addition to typical analytical challenges, perfect agreement

427

between two vastly different technologies such as ELISA and LC-MS/MS is not

428

expected, however correlation of the data does support validation of surrogate peptides

429

representative of intact proteins. Nine individual plants were analyzed for AAD-12,

430

2mEPSPS, and PAT protein levels by ELISA and LC-MS/MS (Figure 7). The LC-

431

MS/MS results were consistently higher likely due to the detection of native and

432

denatured forms of the protein, however the data demonstrated both results were

433

comparable for AAD-12 and PAT. Further work is needed for 2mEPSPS to understand


Page 21 of 43


Page 21 434

the larger than expected difference between ELISA and LC-MS/MS results which in

435

addition to detecting both native and denatured forms of the protein, can be explained by

436

the aforementioned surface absorption of reference standard peptides to labware or a

437

larger portion of 2mEPSPS population in a denatured form which would not be detected

438

by ELISA37.

439

This research demonstrates a multiplex format specifically identifying three proteins in a

440

single injection with high confidence. The signature peptides defined in the multiplex

441

LC-MS/MS method were validated to represent intact proteins as the accuracy of target

442

peptide mean recovery values from recombinant AAD-12, 2mEPSPS, and PAT proteins

443

fortified into three different types of matrices fell between 70-123% across different

444

analysts, laboratories, and instrumentation and data correlated well with ELISA. The

445

ease of transfer of the method with reproducible results and the ability to quantify

446

multiple proteins from a single injection make LC-MS/MS a suitable technology to

447

quantify transgenic proteins in GE crops.

448

Author Information:

449

Corresponding Author

450

Ryan Hill, Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, Indiana 46268,

451

United States. Phone: (317) 337-4864. Email: [email protected].

452

Notes:

453

The authors declare the following competing financial interest(s): RCH, TJO, GS, and BS

454

are employees of Dow AgroSciences LLC, a wholly owned subsidiary of The Dow

455

Chemical Company, which develops transgenic crops and produces insecticides,



Page 22 of 43

Page 22 456

herbicides, and fungicides for agricultural applications. JE and CC are employed by

457

Critical Path Services LLC which performed the independent method assessment under

458

contract from Dow AgroSciences LLC.

459

Acknowledgement:

460

We thank Jeff Gilbert and John Lawry for helpful comments and discussions as well as

461

the Dow AgroSciences immunochemistry group for the ELISA data generated in this

462

study. Enlist E3™ soybeans were jointly developed by Dow AgroSciences and M.S.

463

Technologies L.L.C.

464

Supporting Information: Protein sequences, possible tryptic peptides, selectivity

465

assessment during peptide selection, method validation data (including extraction

466

efficiency, recovery, and peptide stability), independent method assessment recoveries,

467

and ELISA vs. LC-MS/MS results (Figure 7). This material is available free of charge

468

via the Internet at http://pubs.acs.org.

469

References:

470

1. Juma, C.; Gordon, K. Taking Root: Global Trends in Agricultural Biotechnology.

471

Science, Technology, and Globalization Project; Discussion Paper 2014-07,

472

Belfer Center for Science and International Affairs, Harvard University:

473

Cambridge, MA,

474

(http://belfercenter.ksg.harvard.edu/publication/24899/taking_root.html)

475

(Accessed March 18, 2015).

476

2. Westcott, P.; Trostle, R. Long-Term Prospects for Agriculture Reflect Growing

477

Demand for Food, Fiber, and Fuel. United States Department of Agriculture,


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(http://www.ers.usda.gov/amber-waves/2012-september/long-term-prospects-for-

479

agriculture.aspx#.VOeTB3zF98E) (Accessed March 18, 2015).

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3. Naranjo, S. Impacts of Bt Transgenic Cotton on Integrated Pest Management. J. Agric. Food Chem. 2011, 59, 5842-5851. 4. Brookes, G. Weed Control Changes and Genetically Modified Herbicide Tolerant Crops in the USA 1996-2012. GM Crops & Food, 2014, 5:4, 321-332. 5. Lepping, M.D.; Herman, R.A.; Potts, B.L. Compositional Equivalence of DAS-

485

444Ø6-6 (AAD-12 + 2mEPSPS + PAT) Herbicide-Tolerant Soybean and

486

Nontransgenic Soybean. J. Agric. Food Chem. 2013, 61, 11180-11190.

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6. Dryzga, M.D.; Yano, B.L.; Andrus, A.K.; Mattsson, J.L.; Evaluation of the Safety

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and Nutritional Equivalence of a Genetically Modified Cottonseed Meal in a 90-

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Day Dietary Toxicity Study in Rats. Food and Chemical Toxicology, 2007, 45,

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1994-2004.

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7. Global Knowledge Center on Crop Biotechnology: Stacked Traits in Biotech Crops,

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ISAAA Pocket K No. 42, 2013,

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(http://www.isaaa.org/resources/publications/pocketk/42/).

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8. Nicolia, A.; Manzo, A.; Veronesi, F.; Rosellini, D.; An Overview of the last 10

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years of genetically engineered crop safety. Crit Rev Biotechnol, 2013, 1-12,

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1549-7801.

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9. Shan, G.; Embrey, S. K.; Schafer, B.W.; A highly specific Enzyme-linked

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immunosorbent assay for the detection of Cry1Ac insecticidal crystal protein in

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transgenic WideStrike cotton. J. Agric Food Chem. 2007, 55, 5974-5959.



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10. Shan, G., Embrey, S. K., Herman, R. A. and McCormick, R. W. Cry1F Protein

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not Detected in Soil After Three Years of Transgenic Bt Corn (1507 Corn) Use.

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Environmental Entomology, 2008, 37, 255-262.

503 504 505

11. Shan, G. Immunoassays in Agricultural Biotechnology. John Wiley & Sons, Inc. Hoboken, New Jersey, 2011, 1-4. 12. Lundry, D.R.; Burns, J.A.; Nemeth, M.A.; Riordan, S.G. Composition of Grain

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and Forage from Insect-Protected and Herbicide-Tolerant Corn, Mon 89034 x

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TC1507 x MON 88017 x DAS-59122-7 (SmartStax), Is Equivalent to That of

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Conventional Corn (Zea mays L.). J. Agric. Food Chem., 2013, 61, 1991-1998.

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13. Lipton, C.R.; Dautlick, J.X.; Grothaus, G.D.; Hunst, P.L.; Magin, K.M.; Mihaliak,

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C.A.; Rubio, F.M.; Stave, J.W. Guidelines for the Validation and Use of

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Immunoassays for Determination of Introduced Proteins in Biotechnology

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Enhanced Crops and Derived Food Ingredients. Food and Agricultural

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Immunology, 2000, 12, 153-164.

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14. Grothaus, G. D.; Bandla, M.; Currier, T.; Giroux, R.; Jenkins, G. R.; Lipp, M.;

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Shan, G.; Stave, J. W.; Pantella, V. Immunoassay as an Analytical Tool in

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Agricultural Biotechnology. AOAC International. 2006, 89: 913-928.

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15. Zhou, S.; Song, Q.; Tang, Y.; Naidong, W. Critical Review of Development,

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Validation, and Transfer for High Throughput Bioanalytical LC-MS/MS

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Methods. Current Pharmaceutical Analysis, 2005, 1, 3-14.

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16. Palandra, J.; Finelli, A.; Zhu, M.; Masferrer, J.; Neubert, H. Highly Specific and

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Sensitive Measurements of Human and Monkey Interleukin 21 Using Sequential


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Protein and Tryptic Peptide Immunoaffinity LC-MS/MS. Anal.

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Chem., 2013, 85 (11), 5522–5529.

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17. Sack, C.; Smoker, M.; Chamkasem, N.; Thompson, R.; Satterfield, G.; Masse, C.;

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Mercer, G.; Neuhaus, B.; Cassias, I.; Chang, E.; Lin, Y.; MacMahon, S.; Wong,

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J.; Zhang, K.; Smith, R.E. Collaborative Validation of the QuEChERs Procedure

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for the Determination of Pesticides in Food by LC-MS/MS. J. Agric. Food Chem.,

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2011, 59 (12), 6383-6411.

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18. Chamkasem, N.; Ollis, L.W.; Harmon, T.; Lee, S.; Mercer, G.; Analysis of 136

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Pesticides in Avocado Using a Modified QuEChERS Method with LC-MS/MS

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and GC-MS/MS. J. Agric. Food Chem., 2013, 61(10), 2315-2329.

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19. Gardner, M.S.; Voyksner, R.D.; Haney, C.A. Analysis of Pesticides by LC-

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Electrospray-MS with Postcolumn Removal of Nonvolatile Buffers. Anal. Chem.

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2000, 72, 4659-4666.

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20. Lerch, R.N.; Ferrer, I.; Thurman, E.M.; Zablotowicz, R.M. Identification of

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Trifluralin Metabolites in Soil Using Ion-Trap LC/MS/MS. Liquid

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Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS, Chapter

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17, 2003, 291-310.

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21. Vargo, J.D. Determination of Chloroacetanilide and Chloroacetamide Herbicides

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and Their Polar Degradation Products in Water by LC/MS/MS. Liquid

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Chromatography/Mass Spectrometry, MS/MS and Time of Flight MS, Chapter

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14, 2003, 238-255.

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22. Hu, X.T.; Owens, M.A.; Multiplexed Protein Quantification in Maize Leaves by Liquid Chromatography Coupled with Tandem Mass Spectrometry; An



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Alternative Tool to Immunoassays for Target Protein Analysis in Genetically

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Engineered Crops. J. Agric. Food Chem., 2011, 59, 3551-3558.

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23. Zhang, H.; Liu, Q.; Zimmerman, L.J.; Ham, A.L.; Slebos, R.J.; Rahman, J.;

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Kikuchi, T.; Massion, P.; Carbone, D.; Billheimer, D.; Liebler, D.; Methods for

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Peptide and Protein Quantitation by Liquid Chromatography-Multiple Reaction

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Monitoring Mass Spectrometry. Molecular & Cellular Proteomics, 2011, 10.6.

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24. Zhang, Y.; Fonslow, B.R.; Shan, B.; Baek, M.; Yates, J.R. Protein Analysis by

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Shotgun/Bottom-up Proteomics. Chem. Rev., 2013, 113, 2343-2394. 25. Lesur,A.; Varesio, E.; Domon, B.; Hopfgartner, G. Peptides Quantification by

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Liquid Chromatography with Matrix-Assisted Laser Desorption/Ionization and

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Selected Reaction Monitoring Detection. J. Proteome Res., 2012, 11(10), 4972-

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4982.

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26. Lowenthal, M.S.; Liang, Y.; Phinney, K.; Stein, S.E. Quantitative Bottom-Up Proteomics Depends on Digestion Conditions. Anal. Chem.2014, 86(1), 551-558. 27. Yeung, Y.; Stanley, E.R.; Rapid Detergent Removal from Peptide Samples with

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Ethyl Acetate for Mass Spectrometry Analysis. Curr Protoc Protein Sci, 2010,

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16(12).

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28. Hustoft, Hanne Kolsrud, et al. "A critical review of trypsin digestion for LC-MS based proteomics." Integrative Proteomics (2012): 73.

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29. Broek, I.; Smit, N.; Romijn, F.; Laarse, A. Evaluation of Interspecimen Trypsin

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Digestion Efficiency Prior to Multiple Reaction Monitoring-Based Absolute

566

Protein Quantification with Native Protein Calibrators. J. Proteome Res., 2013,

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12, 5760-5774.


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30. Goebel-Stengel, M.; Stengel, A.; Taché, Y.; Reeve Jr.; J.R.; The importance of

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using the optimal plastic and glassware in studies involving peptides. Anal

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Biochem., 2011, 414 (1), 38-46.

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31. Bernert, J.T.; Turner, W.E.; Pirkle, J.L.; Sosnoff, C.S.; Akins, J.R.; Waldrep,

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M.K.; Ann, Q.; Covey, T.R.; Whitfield, W.E.; Gunter, E.W.; Miller, B.B.;

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Patterson, D.G.; Needham, L.L.; Hannon, W.H.; Sampson, E. J. Development and

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validation of sensitive method for determination of serum cotinine in smokers and

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nonsmokers by liquid chromatography/atmospheric pressure ionization tandem

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mass spectrometry. Clinical Chemistry, 1997, 43(12), 2281-2291.

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32. Cohen Freue, G.V.; Borchers, C.H. Multiple Reaction Monitoring (MRM)

578

Principles and Application to Coronary Artery Disease. Circ Cardiovasc Genet.

579

2012, 5, 00-00.

580

33. Lu, Q.; Zheng, X.; McIntosh, T.; Davis, H.; Nemeth, J.F.; Pendley, C.; Wu, S.;

581

Hancock, W.S. Development of different analysis platforms with LC-MS for

582

pharmacokinetic studies of protein drugs. Anal. Chem., 2009, 81(21), 8715-8723.

583 584 585

34. Keith, L.H., Crummett, W., Deegan, J., Libby, R.A., Taylor, J.K., Wentler, G., 1983. Principles of environmental analysis. Analytical Chemistry 55, 2210-2218. 35. Addona, T.A.; Abbatiello, S.E.; Schilling, B.; Skates, S.J.; Mani, D.R.; Bunk,

586

D.M; Spiegelman, C.H.; Zimmerman, L.J.; Ham, A.J.; Keshishian, H.; Hall, S.C.;

587

Allen, S.; Blackman, R.K. Borchers, C.H.; Buck, C.; Cardasis, H.L.; Cusack,

588

M.P.; Dodder, N.G.; Gibson, B.W.; Held, J.M.; Hiltke, T.; Jackson, A.; Johansen,

589

E.B.; Kinsinger, C.R.; Li, J.; Mesri, M.; Neubert, T.A.; Niles, R.K.; Pulsipher,

590

T.C.; Ransohoff, D.; Rodriguez, H.; Rudnick, P.A.; Smith, D.; Tabb, D.L.;



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Page 28 591

Tegeler, T.J.; Variyath, A.M.; Vega-Montoto, L.J.; Wahlander, A.; Waldemarson,

592

S.; Wang, M.; Whiteaker, J.R.; Zhao, L.; Anderson, N.L.; Fisher, S.J.; Liebler,

593

D.C.; Paulovich, A.G.; Regnier, F.E.; Tempst, P.; Carr, S.A. Multi-site

594

assessmentof the precision and reproducibility of multiple reaction monitoring-

595

based measurements of proteins in plasma. Nat Biotechnol. 2009; 27: 633-641.

596

36. Aebersold, R.; Burlingame, A.; Bradshaw, R.A. Western Blots versus Selected

597

Reaction Monitoring Assays: Time to Turn the Tables? Mol Cell Proteomics,

598

2013, 9, 2381-2382.

599 600

37. Shan, G. Immunoassays in Agricultural Biotechnology. John Wiley & Sons, Inc. Hoboken, New Jersey, 2011, 197-198.

601

Figure Captions:

602

Figure 1 –Example extraction buffer evaluation for leaf tissue using (A) PBST,

603

(B) 50 mM Tris(hydroxymethyl)aminomethane hydrochloride, or (C) 50 mM ammonium

604

bicarbonate. 15 mg of soybean leaf tissue containing AAD-12, 2mEPSPS, and PAT

605

proteins was extracted with each respective buffer, digested and analyzed by LC-MS/MS.

606

Peptide response from the overnight digest was plotted (peak area ratio) versus individual

607

extraction buffers for each protein. The optimized amount of tissue per volume buffer

608

and buffer choice for the multiplex LC-MS/MS method was 15 mg lyophilized tissue per

609

1.5 mL PBST.

610

Figure 2 – Time-course digestion efficiency evaluation for a) AAD-12, b) 2mEPSPS,

611

and c) PAT. Digestion was monitored (peak area ratios plotted versus digestion time) for

612

individual replicates and quenched with acid at 4, 10, and 15 hour time points to simulate

613

an overnight digestion. An enzyme-substrate ratio of 1:75 used in the method


Page 29 of 43


Page 29 614

demonstrated digestion completeness at the 12 hour digestion time as longer digestion

615

time did not improve detection and the signal was stable at time points representative of

616

an overnight digestion.

617

Figure 3: Example calibration curves for a) AAD-12 (AAYDALDEATR, 598.3/704.4),

618

b) 2mEPSPS (EISGTVK, 367.2/404.3), and c) PAT (TEPQTPQEWIDDLER,

619

928.9/813.9) from two separate multiplex analyses performed on separate days. The

620

calibration curve termed ‘α’ was prepared in PBST and the calibration curve termed ‘β’

621

was prepared in digested soybean control leaf tissue. Good linearity was observed for

622

AAD-12 and PAT in both analysis formats (Figure 3a and 3c). 2mEPSPS observed

623

peptide loss due to surface absorption to lab materials during sample preparation

624

affecting both signal intensity and linearity (Figure 3b). Linearity of the 2mEPSPS

625

calibration curve was recovered when prepared in digested control tissue.

626

Figure 4: Typical total ion chromatogram (TIC) of a 250 ng/mL peptide standard to

627

demonstrate the detection of AAD-12, 2mEPSPS, and PAT quantitation and confirmation

628

surrogate peptides in a single analysis.

629

Figure 5: a) Summary of extraction efficiency for 3 biological replicates of leaf, root,

630

and seed tissues expressing AAD-12, 2mEPSPS, and PAT serial extracted 5 times each

631

with the equation EE = ቀSum of peptide recovered from all extractionsቁ x 100) applied; b) Example

632

western blot confirmation for extraction efficiency, 2mEPSPS ~47.5 kD (PAT and AAD-

633

12 western blots can be found in supporting data). An immunoreactive band of the

634

expected 47.5 kD size is observed in lanes 10, 11, 12, 13, and 14 representing the

635

2mEPSPS recombinant protein standard at concentrations of 2000, 1000, 500, 100, and

636

50 ng/mL. Some cross reactivity with non-target plant proteins was observed at lower

Amount of peptide recovered in 1st extraction



Page 30 of 43

Page 30 637

molecular weights, however the 47.5 kD immunoreactive band is detected in the first

638

extraction represented in lanes 1 (leaf), 4 (root), and 7 (seed). No immunoreactivity is

639

observed in lanes 2 (leaf), 5 (root), and 8 (seed) representing the 5th serial extraction or in

640

lanes 3 (leaf), 6 (root), and 9 (seed) representing the pelleted tissue after the 5th serial

641

extraction as evidence supporting effective extraction.

642

Figure 6: Example specificity assessment for AAYDALDEATR during AAD-12

643

peptide selection; a) control tissue, b) tissue containing AAD-12, c) digested recombinant

644

AAD-12 protein standard, d) digested recombinant 2mEPSPS standard, e) digested

645

recombinant PAT protein standard. The AAYDALDEATR peptide (0.90 min) is present

646

in tissue expressing AAD-12 and the recombinant protein standard and absent in digested

647

control leaf tissue, 2mEPSPS and PAT recombinant protein standards.

648

Figure 7: Results (ng/mg) for a) AAD-12, b) 2mEPSPS, and c) PAT proteins in leaf

649

tissue from nine individual soybean plants analyzed by surrogate peptide LC-MS/MS and

650

ELISA.


Page 31 of 43


Page 31 651

Table 1: Multiplex LC-MS/MS method information for the quantitation of AAD-12,

652

2mEPSPS, and PAT proteins from soybean tissues.

Protein

AAD-12

2mEPSPS

Peptide

AAYDALDEATR** AAYDALDEATR* †

AAYDALDEATR

IGGGDIVAISNVK EISGTVK** EISGTVK* EISGTVK

§

† §

PAT

DVASWR TEPQTPQEWIDDLER** TEPQTPQEWIDDLER* †

TEPQTPQEWIDDLER

§

SVVAVIGLPNDPSVR

Peptide Protein Collision Molecular Molecular Energy Weight Weight (V) (Da) (kD)

Charge State

MS/MS Transition (m/z )

(+2) (+2) (+2)

598.3/704.4 598.3/591.3 603.3/601.3

26 32 32

1195 1195 1205

(+2) (+2) (+2) (+2)

621.9/631.4 367.2/491.3 367.2/404.3 371.2/499.3

29 18 18 18

1242 733 733 741

(+2) (+2) (+2) (+2)

367.2/519.3 928.9/1300.6 928.9/813.9 933.9/818.9

15 52 38 38

733 1857 1857 1867

(+2)

761.9/784.4

33

1522

*Quantitation peptide, MS/MS transition used for quantitation. **Quantitation peptide, MS/MS transition used for confirmation. †Heavy labeled peptide internal standard.

653

§Confirmation peptide, used to confirm digestion completeness.


32

47.5

21


Page 32 of 43

Page 32 654

Table 2: AAD-12, 2mEPSPS, and PAT quantitative range comparison for

655

individual ELISA and multiplex LC-MS/MS methods.

656

LC-MS/MS Protein LOQ ULOQ (ng/mg) (ng/mg) AAD-12 6.60 165.00 2mEPSPS 12.20 305.00 PAT 6.50 163.00

ELISA LOQ ULOQ (ng/mg) (ng/mg) 1.00 10.00 8.00 200.00 0.12 0.60


Page 33 of 43


Page 33 657

Table 3: Spike recovery results of recombinant AAD-12, 2mEPSPS, and PAT

658

proteins fortified into control soybean leaf, root, and seed tissues performed over

659

multiple days.

Protein Protein Tissue Spike Meana Std ng/mg ng/mg Dev.

660

Day 1

Day 2 a

Range ng/mg

CV Meana Std % ng/mg Dev.

Recovery Across Days a

Range ng/mg

CV Meanb Rangeb CV N % % % %

LOQ c

Std Dev. (3X, 10X)

6.60 7.03 0.10 6.96-7.15 1 6.30 0.73 5.46-6.75 12 101 83-108 9 6 0.61 Leaf 33.00 34.45 0.67 33.74-35.08 2 37.31 2.03 35.88-39.63 5 109 102-120 6 6 (1.83, 6.10) 165.00 162.01 6.69 154.51-167.36 4 171.56 11.90 157.99-180.22 7 101 94-109 6 6 6.60 6.07 0.81 5.38-6.96 13 5.85 0.36 5.44-6.11 6 90 82-105 10 6 0.57 AAD12 Root 33.00 38.56 1.17 37.76-39.90 3 38.29 0.97 37.49-39.36 3 116 114-121 3 6 (1.71, 5.70) 165.00 200.66 1.64 199.23-202.44 1 203.87 9.36 196.82-214.49 5 123 119-130 3 6 6.60 6.31 0.16 6.16-6.48 3 7.31 0.05 7.26-7.36 1 103 93-112 8 6 0.56 7 103 97-112 5 6 Seed 33.00 33.65 1.01 32.94-34.81 3 34.63 2.41 32.13-36.95 (1.68, 5.60) 165.00 150.67 8.34 141.92-158.53 6 162.90 4.76 157.46-166.29 3 95 86-101 6 6 12.20 13.09 0.95 12.51-14.19 7 13.20 1.01 12.12-14.13 8 108 99-116 7 6 0.88 Leaf 61.00 59.29 3.11 55.73-61.43 5 68.15 5.37 62.53-73.23 8 104 91-120 10 6 (2.64, 8.80) 305.00 294.42 11.63 281.24-303.27 4 312.35 4.25 307.81-316.23 1 99 92-104 4 6 12.20 13.28 2.13 10.82-14.52 16 10.87 3.55 6.87-13.67 33 99 56-119 24 6 2.94 2mEPSPS Root 61.00 63.03 3.94 58.91-66.75 6 64.22 16.04 49.06-81.00 25 104 80-133 16 6 (8.82, 29.40) 305.00 291.83 12.16 284.48-305.87 4 312.35 24.00 287.72-335.68 8 99 83-110 7 6 12.20 13.26 1.37 12.38-14.48 10 10.54 3.40 6.74-13.28 32 98 55-122 23 6 2.76 9 92 85-105 8 6 Seed 61.00 53.22 2.29 51.71-55.86 4 58.49 5.19 54.24-64.28 (8.28, 27.60) 305.00 266.55 2.45 263.74-268.28 1 282.97 5.24 277.35-287.72 2 90 86-94 4 6 6.50 6.58 0.09 6.48-6.65 1 5.95 0.43 5.48-6.34 7 96 84-102 7 6 0.45 Leaf 32.50 33.93 1.55 32.57-35.62 5 29.36 1.53 27.82-30.87 5 97 86-110 9 6 (1.35, 4.50) 162.50 147.77 5.10 142.49-152.67 3 136.83 3.39 133.44-140.23 2 88 82-94 5 6 6.50 6.04 0.21 5.81-6.22 3 6.08 0.31 5.72-6.29 3 93 88-97 4 6 0.24 PAT Root 32.50 31.06 3.75 27.14-34.60 12 36.53 3.44 32.57-38.79 12 104 84-119 13 6 (0.72, 2.40) 162.50 151.16 5.80 144.75-156.06 4 177.54 4.08 174.15-182.07 4 101 89-112 9 6 6.50 5.49 0.16 5.37-5.68 3 5.42 0.58 4.90-6.05 11 84 75-93 7 6 0.38 Seed 32.50 37.54 1.67 36.53-39.47 4 30.01 1.16 29.18-31.32 4 104 90-121 13 6 (1.14, 3.80) 162.50 160.20 5.34 156.06-166.24 3 142.86 11.99 130.05-153.8 8 93 80-102 8 6 a Results represent detected surrogate peptides converted into protein concentrations represented as dry tissue weight (ng/mg). b Accuracy was indicated as percentage peptide recovery converted into protein concentrations from recombinant protein fortifications. c LOD or LOQ statistical confirmation using 3× or 10× the standard deviation from the LOQ detected response.



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Page 34 661

Table 4: Digested surrogate peptide stability for AAD-12, 2mEPSPS, and PAT

662

evaluated over 5 days. a

AAD-12 Average Recovery Results (%) Leaf Root Seed Fortification N Day 0 Day 5 Day 0 Day 5 Day 0 Day 5 LOQ 3 106 76 92 103 111 102 Mid-Range 3 104 111 117 113 105 105 High Range 3 98 101 122 113 99 100 a

2mEPSPS Average Recovery Results (%) Leaf Root Seed Fortification N Day 0 Day 5 Day 0 Day 5 Day 0 Day 5 LOQ 3 107 99 109 86 109 112 Mid-Range 3 97 98 103 100 87 102 High Range 3 96 86 96 99 87 95 a

PAT Average Recovery Results (%) Leaf Root Seed Fortification N Day 0 Day 5 Day 0 Day 5 Day 0 Day 5 LOQ 3 101 92 93 79 83 113 Mid-Range 3 104 100 96 92 92 88 High Range 3 91 87 93 97 88 83 a

663

AAD-12, 2mEPSPS, and PAT recombinant proteins fortified into leaf, root, and seed control tissues analyzed on Day 0. Fortified tissues and reference peptide standards were stored at 4°C and reanalyzed on Day 5.


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Page 35 664

Table 5: Independent laboratory accuracy and precision assessment for the

665

multiplex LC-MS/MS detection of AAD-12, 2mEPSPS, and PAT proteins in

666

soybean tissues.

Protein

Tissue

Leaf

AAD12

Root

Seed

Leaf

2mEPSPS

Root

Seed

Leaf

PAT

Root

Seed

Recoverya

Protein Spike ng/mg

Mean (%)

Range (%)

CV (%)

6.60 33.00 165.00 6.60 33.00 165.00 6.60 33.00 165.00 12.20 61.00 305.00 12.20 61.00 305.00 12.20 61.00 305.00 6.50 32.50 162.50 6.50 32.50 162.50 6.50 32.50 162.50

83 87 92 70 90 87 100 74 78 80 75 85 69 76 96 74 65 74 70 77 80 107 86 89 75 62 63

70-97 67-98 81-110 60-81 86-97 86-88 85-127 71-78 73-83 74-87 68-78 73-91 65-71 72-83 94-99 62-81 62-68 71-77 64-75 73-83 72-90 102-116 77-98 84-97 71-79 57-67 60-66

17 20 17 15 7 1 24 5 6 8 8 12 5 8 3 14 5 4 7 7 11 7 13 8 6 8 4

a

667

Interlaboratory method evaluation performed by Critical Path Services. Results indicate recovery (%) of recombinant proteins (detected surrogate peptide converted into protein concentrations) fortified into soybean leaf, root, and seed tissues at the limit of quantitation, mid-range and high range concentrations.



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Figure 1:

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Figure 2:

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Figure 3:

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Figure 4:

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Figure 5:

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Figure 6:

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Figure 7:

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228x77mm (113 x 113 DPI)


Development and Validation of a Multiplexed Protein Quantitation

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