Analysis of Allergenic Proteins using LC/MS- identifying peptide markers to peanut and

tree nuts

Robert D. Voyksner1, Jerry Zweigenbaum2 and Jennifer Sealey-Voyksner3

1 LCMS Limited: 1502 West NC-54, Suite 504, Durham, NC, USA 277072 Agilent Technologies: 2850 Centerville Road, BL3-2 3L11 Wilmington, DE, USA 198083 ImmunogenX: 1502 West NC-54, Suite 504, Durham, NC, USA 27707

• New marker peptides have been identified in peanut and various tree nuts (almond, pecan, cashew, walnut, hazelnut, pine nut, brazil nut, macadamia nut and pistachio).

NOVEL ASPECT

• This presentation will discuss the development of an analytical LC/MS-based methodology for the identification of marker peptides for the purpose of qualitative determination of the presence of peanut and various tree nuts in native and processed food products.

• LC-MS is a sensitive and versatile analytical technique which is being used effectively by many researchers to study allergenic tree nut and peanut proteins. This presentation will discuss how accurate mass and MS/MS data obtained from LC-QTOF mass spectrometric analyses of raw and processed food, permitted the identification of multiple marker peptides for each nut representing peanut and nine different tree nuts.

• Data shown will describe the methods, tools and data interpretation processes that allowed the most useful marker peptides to be identified. Future work will involve the incorporation of these markers into a quantitative LC-MS/MS method allowing for the analysis of complex food matrices for purposes of measurement of trace levels of such allergens. This work has important implications for manufacturing certification and food labeling purposes.

PURPOSE

Important issues for consideration using LC/MS to study allergens

> LC/MS versus ELISA

- LC/MS is considered by some researchers as an emerging technique !!

- ELISA has major drawbacks in allergen analysis compared to LC/MS

- FDA endorses ELISA; no validated LC/MS yet

- commercial availability

Chemical and Engineering News, October 12, 2012

• Tree nuts and peanuts constitute a nutritious part of a healthy diet for most people and are commonly used as healthful ingredients in processed food products. As such, the consumption of nuts and prevalence of nuts in food products have both been steadily increasing, worldwide.

• Unfortunately, peanut and tree nuts are among the most common foods to cause allergenic reactions, seriously affecting the quality of the lives of millions of children and adults. They are responsible for the most serious allergenic reactions and food-allergy related deaths. The only means for those who suffer allergenic reactions to peanut and tree nuts to remain healthy, is strict avoidance of these allergens. Therefore, it is in the consumer’s best interest to know what is in the products they will be eating, and that they are free from potential allergens.

• Peanuts and tree nuts are sometimes present in trace amounts in food products, as unwanted ingredients, due to processing, contamination or other such processes. Sensitive, accurate and reliable analytical methods must be available for the detection of trace levels of allergens for food labeling and manufacturing certification purposes.

• Food products undergo many processing procedures including heating, pressurization and sterilization. These lead to modification of proteins.

• Care must be taken in the choice of marker peptides to ensure their presence in both native and processed states.

INTRODUCTION and BACKGROUND

• Current research has shown that LC-MS can be used effectively to identify tryptic fragments of allergenic tree nut and peanut proteins. This presentation will discuss the characterization of some tree nut and peanut proteins using accurate mass LC-MS and LC-MS/MS to identify products of various digestive enzymes under conditions that mimic the human digestive process. (Data shown here is limited only to tryptic digestion products).

• Marker peptides for each nut were carefully chosen as those that could be detected in both raw and processed food.

• The methodology was tested on a variety of foods to validate the presence/absence of the various peptide markers in both native and processed foods. Future work will continue to develop this LC-MS method into a quantitative method. These efforts will pave the way for eventual commercial application as a service to the food industry, as an economic means to identify the presence/absence of a variety of nuts in food with a single assay.

The Basics of Foodomics

Proteins are components in food stuffs and can be used to specifically identify a food product. These proteins are often the direct reason for allergenic reactions.

Digestions of these proteins can yield specific marker peptides which can identify the food protein.

LC/MS/MS and High resolution MS analysis can specifically identify these peptides for measuring the presence and levels of the food stuff in a sample.

The key is to find peptides that are not modified from food processing (heating, fermentation, hydrolysis) to represent food products to achieve minimal false negatives and to select peptides that are unique to that food product ( vs other food products) to minimize false positives.

- Trypsin digestion - LC/MS analysis - database search

Peanuts and tree

nuts

Select most abundant peptides that represent that nut)

Confirm identity with synthetic standard

Abundances <20% between processed and native state

Absent in all other plantsPotential marker peptide for

peanuts and tree nuts

Sequence search in databases shows no matches with other

plants

Work flow for the discovery of marker

peptides forTree nuts and peanuts

1. Initial work involved characterization of the protein profiles of raw versus roasted peanut and tree nut samples; various extraction and fractionation conditions were investigated.

2. Proteins from raw and roasted nuts were extracted, then proteolytically digested in-vitro, using conditions and enzymes that model the human digestion process (pepsin, trypsin and chymotrypsin) – data shown here is trypsin only

3. The peptide digests were analyzed using accurate mass LC-MS and LC-MS/MS; potential representative peptides were identified and sequenced through MS/MS database searches.

4. Various food samples were analyzed for the presence/absence of each nut by qualitatively screening the samples for the representative marker peptides.

METHODSOverview of experiments

Sample preparation:• 30g of the each nut (raw and roasted – 30min @350˚C) were ground to a flour and homogenized: peanut, almond, pecan, cashew, walnut, hazelnut, pine nut, brazil nut, macadamia nut and pistachio

Protein extraction:• proteins from each sample were extracted (2 hours @50˚C) using the following 3 conditions:

1) 0.01M HCl (pH 2.5-3); 2) 50mM TRIS/HCl (pH 7.5); 3) 50mM phosphate (pH 8)

Off-gel protein fractionation:• samples were fractionated using 2 gels (pH 3-10 and 4-7) for 64KVh (about 24 hrs) using an Agilent Off-Gel Fractionator Model 3100

WCX fraction collection:• samples were also collected via WCX (Agilent Bio WCX NP 1.7μm nonporous 4.6mm x 50mm) with fractions collected every 1 min. using an Agilent 1260 Fraction Collection System

LC separation:• all protein fractions were analyzed using SEC (Agilent BioSEC 3, 3μm, 100Å, 4.6 x 150mm) and RP (Agilent Poroshell 300 2.1x75mm, 2.7μm, 300Å)

LC-QTOF:Agilent 6530 High Definition Q-TOF with 1290 Infinity LC system; operated in +ESI mode

1. Protein extraction and fractionation conditions

• extracted protein samples were enzymatically digested with trypsin; protein extracts were centrifuged, and 200μL of supernatant analyzed by LC-MS:

LC-MS Conditions for Peptide Discovery and Identification

2. Enzymatic digestion

RPLC separation:column: Poroshell 120 SB C18 2.1x50mm; 0-40% B in 60 or 90 min then to 70% B in 15minA= 95/5 water/ACN + 0.025% TFA; B= 95/5 water/ACN + 0.025% TFA; flow rate 0.25 ml/min

LC-QTOF:Agilent 6530 High Definition Q-TOF with 1290 Infinity LC system; operated in +ESI mode

Database searches:MS/MS spectra were searched in green plant databases (i.e. SwissProt and NCBI)

Sample preparation:• 30g of various food samples were ground to a powder and homogenized• proteins were extracted using 50mM TRIS/HCl (pH 7.5)• extracts were digested with trypsin• digested extracts were analyzed by LC/MS

RPLC separation:column: Poroshell 120 SB C18 2.1x50mm; 0-40% B in 60 or 90 min then to 70% B in 15minA= 95/5 water/ACN + 0.025% TFA; B= 95/5 water/ACN + 0.025% TFA; flow rate 0.25 mL/m

LC-QTOF:Agilent 6530 High Definition Q-TOF with 1290 Infinity LC system; operated in +ESI mode

3. Analysis of processed food products

Comparison of protein molecular weights between roasted and unroasted peanuts

1195811940

2450224484

61743

58419 5805258250

51784

Comparison of protein molecular weights between roasted and unroasted peanuts

3x10

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1

Counts vs. Mass-to-Charge (m/z)

400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800

+ESI Scan (17.759-17.875 min, 15 Scans) Frag=160.0V peanut-r-2.d

1221.9910 2421.9135704.5148

1062.6772

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8

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Counts vs. Mass-to-Charge (m/z)

400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800

+ESI Scan (17.753-17.877 min, 16 Scans) Frag=160.0V peanut-ur-2.d

1221.9910

2421.91341076.2874

429.0611

MW 58.4 kDa

MW ?

> the observed protein profiles and tryptic peptide intensities of raw nuts were different (sometimes significantly) between the raw and the roasted state

Important issues for consideration using LC/MS to study allergens

Changes in major proteins in various nut extracts between roasted and unroasted (native)

Nut Roasted (Da) Native (Da)Almond 60225* 53298

Brazil Nut 51011 49732Cashew 50192 50192Hazelnut 52741* 51601

Macadamia Nut 79383* 57996Peanut 58250* 55380Pecan 58069* 51228

Pine Nut 70319* 48012Walnut 61979* 33093

Nut Roasted (Da) Native (Da)Almond 60225* 53298

Brazil Nut 51011 49732Cashew 50192 50192Hazelnut 52741* 51601

Macadamia Nut 79383* 57996Peanut 58250* 55380Pecan 58069* 51228

Pine Nut 70319* 48012Walnut 61979* 33093

* more than 1000 spread in molecular weights

41 new peptides found in roasted peanuts

Tryptic peptides found in roasted vs. unroasted peanuts

Comparison of intensities of a tryptic peptide marker found in hazelnut

roasted

unroasted

LLSGIENFR

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

unroasted

roasted

Differences in Peptide intensity between roasted and unroasted Hazelnuts

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Counts vs. Acquisition Time (min)

4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66

+ESI EIC(564.8130-564.8330, 628.3632-628.3832 ...) Scan p2 ur-t.d

33.59 29921608.9519.98 17922350.12

12.09 320256.21

12

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Counts vs. Acquisition Time (min)

4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66

+ESI EIC(564.8130-564.8330, 628.3632-628.3832 ...) Scan p2 r-t.d

33.69 23376244.0520.17 14306041.24

12

Examples of three 3 peanut marker peptides detected in food

unroasted peanut

roasted peanut

SFNLDEGHALRGTGNLELVAVR

TANDLNLLILR

3x10

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Counts vs. Mass-to-Charge (m/z)

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

Cpd 18: 20.025: +ESI Product Ion (19.833, 19.847, 19.862, 19.876 ... min, 8 Scans) Frag=150.0V CID@15.1 (629.8132[z=2] -> **) p2 ur-t.d

629.8130

207.1129

797.3891 512.7619

87.0554 349.1498

682.3615 1024.5160 910.4722 462.2331 288.2024

MS/MS spectra of peanut peptide SFNLDEGHALR

y9y8

y7

y6

y9+2

b3

a2

b1

(M+2H)+2

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Counts vs. Acquisition Time (min)

4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66

+ESI EIC(564.8130-564.8330, 628.3632-628.3832 ...) Scan nutter butter-t.d

33.92 6006063.66

20.27 2540172.55

12

peanut butter snack

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0.75

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Counts vs. Acquisition Time (min)

4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66

+ESI EIC(564.8130-564.8330, 628.3632-628.3832 ...) Scan proten bar-t.d

34.00 3940585.45

20.39 1052786.08

12

protein bar

Comparison of intensities of tryptic peptide markers found in roasted and unroasted almonds

Increases by roastingDecreases

by roasting

ALPDEVLQNAFRALPDEVLQNAFR

GNLDFVQPPR

LKENIGNPER

ISTLNSHNLPILRTEENAFINTLAGR

Nut RT (min) Sequence Accurate Mass (m/z) Charge State MS/MS mass (m/z) Fragment type Protein MW (kDa) Cross correlation with other nuts

Peanut 18.1 SPDIYNPQAGSLK 695.3544 2 977.5047 y9 glycinin [Arachis hypogaea] 58.315 Almonds and Pistachio814.4402 y8

33.9 WLGLSAEYGNLYR 771.3926 2 1242.6112 y11 glycinin [Arachis hypogaea] 60.974 Pecans1072.5051 y9

Almond 10.4 LKENIGNPER 585.3182 2 928.447 y8 prunin [Prunus dulcis] 62.979 Cashew799.4059 y7

Pecan 33.9 WLGLSAEYGNLYR 771.3926 2 1242.6112 y11 glycinin [Arachis hypogaea] 60.974 Peanuts1072.5051 y9

42.4 AFSWEVLEAALK 682.3693 2 1145.6207 y10 glycoprotein [Corylus avellana] 50.825 Hazelnuts (roasted), Walnuts872.5093 y8

36.4 VFSNDILVAALNTPR 815.4545 2 954.5715 y9 vicilin [Juglans regia] 69.947 Walnuts742.4206 y7

Pistachio 13.3 NSFDISR 419.707 2 490.2613 y4 11S globulin [Pistacia vera] 56.431 Cashew637.3294 y5

Walnut 34 GDIFVIPAGHPIAITASQNENLR 1217.6524 2 1426.7677 y13 vicilin [Juglans nigra] 55.641 Pecan1789.9439 y17

Hazelnut 25.5 LLSGIENFR 524.7942 2 822.4087 y7 glycoprotein [Corylus avellana] 50.825 Hazlenut (roasted)735.376 y6

23.6 WLQLSAER 501.7731 2 703.3717 y6 11S globulin [Corylus avellana] 59.092 Walnuts, Pistachios, Brazil nuts and Cashews575.3142 y5

Cross Correlation – homologous sequences

Discovered marker peptides for peanuts and tree nutsNut (Latin name)

Selected peptide Sequence MS/MS sequence Intensity Ratio Peak area (x 106) Accurate Mass Mass error Protein Name Protein Accession # Protein Cross activity with other nuts NCBInr search results

marker ( ID) Score unroasted/roasted (m/z) (mmu) MW (KDa) based on LC/MS studiesPeanut p1 SFNLDEGHALR 22.8 1.14 135 629.8133 0.2 conarachin ara h1 46560478 66.973 Allergen Ara h 1

(arachis hypogaea)VLLEENAGGEQEER 24.6 1.02 123 786.8799 0.6 conarachin ara h1 46560478 66.973 Ara h1 allergen

p2 GTGNLELVAVR 17.8 1.11 93 564.823 0.2 conarachin ara h1 46560478 66.973 Ara h1 allergen

p3 TANDLNLLILR 21 1.12 330 628.3732 0.2 gly1 9864777 60.477 Ara h2 and 4

SPDIYNPQAGSLK 21.2 1.19 368 695.3544 1.1 gly1 9864777 60.477 Almonds / Pistachio Ara h1, 2 and 4

FNLAGNHEQEFLR 23.3 0.98 89 787.89 0.7 gly1 9864777 60.477 10 hit all peanuts

p4 AHVQVVDSNGDR 20.2 1.08 309 648.3273 0.1 allergen Arah3/Arah4 21314465 61.737 Arah3/Arah4

WLGLSAEYGNLYR 24.1 1.01 186 771.3926 0 gly1 9864777 60.477 Pecans Ara h 3/4

Almond a1 TEENAFINTLAGR 19.5 0.92 93 718.3637 0.1 prunin 460806 63.016 prunin(prunis dulcis)

a2 ISTLNSHNLPILR 24.9 0.94 161 739.4292 0.3 prunin 460806 63.016 prunin

LKENIGNPER 18.9 0.99 42 585.3182 0.4 prunin 460806 63.016 Cashew prunin

GNLDFVQPPR 16.8 1.05 72 571.8021 0.1 prunin 460806 63.016 prunin

Pecan LLQPVNNPGQFR 18.1 1.19 16 691.8823 1.6 vici;in precursor 6580762 69.989 Walnut walnut/pecans(carya illinoinensis)

ATLTFVSQER 17.8 0.84 30 576.3083 2.1 7s Vicilin 157384600 95.167 pecans

pe1 LVGFGINGK 18.1 1.09 161 904.5251 0.4 7s Vicilin 157384600 95.167 Pecan

pe2 QFQEQQER 17.8 1.03 72 546.7578 0.4 7s Vicilin 157384600 95.167 Pecan

Brazil nut b1 LAENIPSR 17.7 1.02 317 450.2516 0.3 2S albumin 8439533 16.909 brazil nuts

(bertholletia excelsa)b2 MAENLPSR 18.7 0.98 393 917.4514 0.5 2S albumin precursor 209738721 16.755 brazil nuts

GGQQILADNVFK 18.9 1.09 203 645.3472 1.2 11s globulin 30313867 52.295 brazil nuts

GETVFDDNLR 17.2 0.96 161 583.2789 1 11s globulin 30313867 52.295 brazil nuts

Cashew c1 QLQQQEQIK 17.7 1.05 123 571.8129 0.1 2s albumin 24473800 16.335 cashew

(anacardium occidentale)c2 ADIYTPEVGR 18.9 1.1 169 560.7861 0.2 allergen Ana o 2 25991543 51.995 cashew

ELYETASELPR 19.9 1 216 654.3287 0.1 2s albumin 24473800 16.335 pitachio

WLQLSVEK 15.3 1.12 135 1002.5628 0.4 allergen Ana o 2 25991543 51.995 cashew

Pistachio pis2 LQELYETASELPR 19.1 1 131 774.9006 1.8 Pis v 1 allergen 2S albumin 110349081 17.289 pitachio(pistacia vera)

pis1 TNGLSQTSQLAGR 19.5 1 76 666.8486 0.1Pis v 2.0101 allergen11S globulin precusor-

Pistacia vera 110349083 56.464 pitachio

NIIEVMEK 15.8 1 72 488.264 0.1 vicilin 133711974 59.772 pitachio

AMISPLAGSTSVLR 21.9 1 27 701.8912 1.8 Pis v 5.0101 allergen11S globulin precusor 171853010 53.928 pitachio

Walnut w1 GEEMEEMVQSAR 23.1 0.92 186 698.2989 0.1 albumin seed storage pro jug r1 1794252 16.373 walnuts(juglans)

GDIFVIPAGHPIAITASQNENLR 21.1 1.15 72 877.7701 0.8 vicilin seed storage prot jug r2 6580762 69.999 pecan/walnut

LLGFGINGENNQR 23.6 0.96 37 716.3722 1.8 vicilin seed storage prot jug r2 31321944 55.674 walnut

w2 QQQQQGLR 15.9 1.07 89 493.2634 1.1 albumin seed storage prot jug r1 1794252 16.373 walnut

Nut (Latin name) Selected peptide Sequence RT (min) Accurate Mass Charge State Collision energy MS/MS mass (m/z) Fragment typemarker ( ID) (m/z) (V)

Peanut p1 SFNLDEGHALR 20 629.8133 2 15.7 797.3891 y7(arachis hypogaea) 682.3615 y6

p1 Int standard SFNLDEGHALR 20 634.8174 2 15.8 807.3973 y7692.3697 y6

p2 GTGNLELVAVR 22.2 564.823 2 14.1 686.4175 y6444.2917 y4

p3 TANDLNLLILR 33.6 628.3732 2 15.7 741.4969 y61083.6504 y9

p3 Int standard TANDLNLLILR 33.6 633.3732 2 15.8 751.5051 y61093.6586 y9

p4 AHVQVVDSNGDR 6.9 648.3273 2 16.2 1087.5488 y10989.4647 y9

Almond a1 TEENAFINTLAGR 28.2 718.3637 2 18.0 891.5026 y8(prunis dulcis) 631.3522 y6

a2 ISTLNSHNLPILR 26.9 739.4292 2 18.5 498.3382 y4725.4668 y7

Pecan pe1 LVGFGINGK 22.1 904.5251 1 36.2 692.3721 y7(carya illinoinensis) 488.2855 y5

pe2 QFQEQQER 14.4 546.7578 2 13.7 689.3171 y5817.3772 y6

Brazil nuts b1 LAENIPSR 11.3 450.2516 2 11.3 359.2031 y3

(bertholletia excelsa) 715.3746 y6

b2 MAENLPSR 10.5 917.4514 1 36.7 586.3291 y5446.1689 b4

Cashew c1 QLQQQEQIK 8.1 571.8129 2 14.3 773.4134 y6

(anacardium occidentale) 645.355 y5

c2 ADIYTPEVGR 16.9 560.7861 2 14.0 821.4138 y7658.3503 y6

Pistachio pis2 LQELYETASELPR 25.7 774.9006 2 19.4 1065.522 y9(pistacia vera) 1307.647.3 y11

pis1 TNGLSQTSQLAGR 13.3 666.8486 2 16.7 947.4897 y9732.3999 y7

Walnut w1 GEEMEEMVQSAR 19.7 698.2989 2 17.5 461.2465 y4(juglans) 820.398 y7

w2 QQQQQGLR 4.6 493.2634 2 12.3 345.2261 y3729.4011 y6

MRM transitions for marker peptides for peanuts and selected tree nuts

Calibration of peanut protein and walnut protein to marker peptide concentration

y = 0.023x - 0.05R² = 0.9992

-5

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ppm peptide

y = 0.0023x - 0.0108R² = 0.9997

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0 200 400 600 800 1000 1200

ppm peptide

ppm walnut

TANDLNLLILR

GEEMEEMVQSAR

Detection of tree nuts in coconut cream cakewalnuts were listed on label but pecans were not

listed+ MRM (812.1 -> 895.0) A-02 corn flour.d

Acquisition Time (m

8 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9 9.1 9.2 9.3 9.4 9.5 9.6

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8.769 min.

+ MRM (812.1 -> 895.0) protein08.d

Acquisition Time (m

8 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9 9.1 9.2 9.3 9.4 9.5 9.6

Counts

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1.58.828 min.

+ MRM (812.1 -> 895.0) A-08 cocunut cream cake.d

Acquisition Time (m

8 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9 9.1 9.2 9.3 9.4 9.5 9.6

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8.777 min.

Walnuts Pecans

Blank

100 ppm std

Sample:237 ppm measured

+ MRM (815.8 -> 841.6) A-02 corn flour.d

Acquisition Time (m

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9.759 min.

+ MRM (815.8 -> 841.6) protein07.d

Acquisition Time (m

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9.836 min.

+ MRM (815.8 -> 841.6) A-08 cocunut cream cake.d

Acquisition Time (m

8.9 9 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 10 10.1 10.2 10.3 10.4 10.5 10.6 10.7

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9.784 min.

Blank

10 ppm std

Sample:26 ppm measured

Qualitative results for peanut and tree nut peptides by LC-MS/MS

Manufctures in Facility thatProduct Tested Nut Ingredients Listed also processes listed nuts Peanuts Almonds Pecans Pine nut Cashews Brazil Hazelnuts Walnuts Macadamia Pistachio Chestnuts CoconutBanana nut muffin almonds, walnuts tree nits

pecan cookie pecans, coconut peanuts, tree nutsbanana crean cake none tree nuts

peanut butter pretzil peanuts none listedgluten free crackers peanuts pecansnutter butter cookie peanuts none listed

macadamia nut cookie macadamia, coconut pecans, almondsnut crisps cashews, pecans, almonds tree nutsprotein bar peanuts, cashews, almonds, brazil none listed

gluten free bar cashews none listedcorn flour none listed none listed

wheat flour none listed none listedsoy flour none listed none listed

quinona flour none listed none listedGF Pasta Quinea none listed none listed

GF Seasoning none listed none listedGF Crackers hazelnuts pecans, almonds,

GF Pasta -made in wh facility none listed none listedChocolate covered Prezels peanuts walnuts

GF Vit C mix none listed none listedCandy corn none listed Peanuts, tree nuts

Instant Oat meal walnuts tree nutsMisco soup cup none listed none listed

Falafil mix none listed peanuts, tree nutscracker jacks peanuts none listed

hot souce none listed none listedcereal walnuts none listed

cracker crisps chestnut none listedchestnut puree chestnut none listed

coconut nut cake almonds, pistachio tree nutsGronola bar none listed peanuts, almomds, pecans

coffee coated almonds almonds peanuts, tree nutsListed ingredientNut listed as posible contamination

Nut not listed as a possible contamination

• the observed protein profiles of raw nuts were different (sometimes significantly) between the raw and the roasted nut

• relative intensities of some digested peptides were sometimes very different between the raw and roasted state

CONCLUSIONS

• resource protein database information is not complete for tree nuts and peanut; leading to discrepancies between observed sequences and theoretical; homologous sequences observed between species is an important criteria in choosing the marker peptides; it was observed that several peptide sequences were found in other nut proteins, thus rendering them poor candidates for specific markers for a given nut (false positives/negatives)

• marker peptides were chosen based on their presence in both raw and processed food, absence in other nuts and non nut-containing products; retention time and MS/MS fragment ion intensity ratios

• the use of multiple peptide markers for each nut better ensures no false positives/negatives

• the LC-QTOF offered several advantages in biomarker peptide discovery:– molecular weights of potential marker peptides could be determined in 1-3 mmu – EICs using <10 mmu windows (centered on the postulated peptide MW) greatly

simplified the spectra allowing faster discovery of marker peptides (as compared to numerous peaks observed in a LC-MS run using a 1 Da window)

– accurate mass and auto MS/MS aided in verifying amino acid composition, sequencing and reduced the number of possible hits in the database search process

• this is a novel methodology (qualitatively determining the presence of 10 nuts in one assay) with the presence of each nut in an unknown native or processed sample verified using at least 2 unique marker peptides; future work will expand the methodology to a LC-MS/MS assay suitable for trace level quantitation

• LCMS Limited:Robert Voyksner: robert_voyksner@lcmslimited.com

• ImmunogenX:Jennifer Sealey-Voyksner: jsealey-voyksner@immunogenx.com

• Agilent Technologies:Jerry Zweigenbaum: j_zweigenbaum@agilent.com

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