manchester institute of biotechnology food allergen analysis: … · 2017-11-14 · victoria lee 1,...

Victoria Lee1, Rebekah Sayers1, Ivona Baricevic-Jones1, Anuradha Balasundaam1, Carol Ann Costello1, Lee

Gethings2, Antonietta Wallace2, Jim Langridge2, Clare Mills1

1Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences and Manchester Institute of Biotechnology,

The University of Manchester; 2 Waters Corporation, Wilmslow UK.

Food allergen analysis: discovery and targeted proteomics

Manchester Institute of Biotechnology

Declaration of interests

Current Funding: UK Food Standards Agency UK Biological and Biotechnological Sciences Research Council UK Medical Research Council UK Innovate European Union NW Lung Centre Charity DBV Technologies In-kind sponsorship of students and collaborations Waters Corporation, Romer Laboratories Ltd, Synergy Health, R-Biopharm, Campden BRI, Fera Spin-out company ReactaBiotech Ltd

There is no accepted cure – so food allergic individuals have to practice food avoidance

• Mandatory labelling of ingredients has helped - but what about all the precautionary labelling?

• A lack of consensus on threshold doses is hampering management of allergens in foods

• Having access to “free-from” foods they can trust is very important for allergic consumers

How low do we need to go?

Immune-mediated Food Allergy

Non-immune-mediated Food Intolerance

IgE mediated

Non-IgE mediated

Enzymatic

Pharmacologic

Undefined

Adverse Reactions to Foods

Toxic Non-Toxic

European Academy of Allergy and Clinical Immunology Classification of Adverse Reactions to Foods

So what triggers an allergic reaction to food? • A molecule called

IgE normally produced to fight parasites

• Protein molecules in foods known as allergens

• These interact to trigger release of histamine from certain kinds of cells

Cupins (11S/7S globulins)

Bet v 1

Prolamins (LTPs, 2S albumins Amylase inhibitors)

Caseins

Tropomyosin

Parvalbumin

as1-, as2-, and b-caseins

K- casein

CMP ‘hairy’ layer

Ca9(PO4)6 cluster

Allergens are almost all proteins!

Dose 1: designed to give the no observed adverse effect level (NOEL) and lowest observed adverse effect level (LOAEL) Dose 9: Equivalent to a daily serving

Dose

no

Mass Protein Matrix

dose (%)

Cumulative

Dose

(mass)

1 6µg 3µg 0.0006 6µg

2 60µg 30µg 0.0006 66µg

3 600µg 300µg 0.06 666µg

4 6mg 3mg 0.06 6.67mg

5 60mg 30mg 3 66.67mg

6 0.12g 600mg 3 0.186g

7 0.6g 0.3g 3 0.786g

8 4.5g 2.2g 3 5.286g

9 6g 3g 3 11.286g

Dosing protocol used for oral food challenges

Ballmer-Weber et al J Allergy Clin Immunol 2015, 135, (4), 964-71.

Dose-response modelling using Lowest Observed Adverse Effect Levels (LOAELs) and interval censoring survival analysis

Ballmer-Weber et al J Allergy Clin Immunol. 2015 ;135(4):964-71.

1e-03 1e+00 1e+03 1e+06

0.0

0.2

0.4

0.6

0.8

1.0

dose (log-scale)

pro

bability

objective symptoms, peanut (50)A

1e-03 1e+00 1e+03 1e+06

0.0

0.2

0.4

0.6

0.8

1.0

dose (log-scale)

pro

bability

objective symptoms, hazelnut (89)B

1e-04 1e-01 1e+02 1e+05

0.0

0.2

0.4

0.6

0.8

1.0

dose (log-scale)

pro

bability

objective symptoms, celery (41)C

1e-04 1e-01 1e+02 1e+05 1e+08

0.0

0.2

0.4

0.6

0.8

1.0

dose (log-scale)

pro

bability

objective symptoms, Fish (33)D

1e-04 1e-01 1e+02 1e+05 1e+08

0.0

0.2

0.4

0.6

0.8

1.0

dose (log-scale)

pro

bability

objective symptoms, Shrimp (27)E

• Dose-response modelling gave a ED10 value of ~3 mg peanut protein depending (log-normal model). This equates to ~12 mg of peanut seed.

• This value is similar to the 12.3 (9.0,

16.8 95% confidence intervals) mg of peanut (median age 7) reported previously (Taylor et al Food Chem 2010)

Voluntary Incidental Trace Allergen Labelling (VITAL) Scientific Expert Panel Proposed Action Levels

Allergen

Reference

dose

(mg Protein)

Action Level (ppm) per

serving size

5 g 50g 250g

Peanut 0.20 40 4.0 0.80

Milk 0.10 20 2.0 0.40

Egg 0.03 6 0.6 0.12

Hazelnut 0.10 20 2.0 0.40

Soy 1.00 200 20.0 4.00

Wheat 1.00 200 20.0 4.00

Tree nuts 0.10 20 2.0 0.40

Mustard 0.05 10 1.0 0.20

Lupine 4.00 800 80.0 16.00

Sesame 0.20 40 4.0 0.80

Shrimp 10.00 2000 200.0 40.00

No VITAL reference dose has been identified for fish and celery due to lack of data

1 2 3 4 5

02

46

81

0

Egg data, concentration=3ppm

kit

pp

m e

gg

wh

ite

pro

tein

Kit=1, mean= 1.41ppm Kit=2, mean= 2.39ppm Kit=3, mean= 2.48ppm Kit=4, mean= 2.58ppm Kit=5, mean= 2.91ppm

Determination of egg in chocolate dessert matrix by ELISA

Johnson et al Food Chem 2014, 148, 30-6

• The chocolate dessert is designed ot be consumed in 100g portions

• VITAL reference dose would around 0.3 mg/Kg which ELISAs would struggle to detect

How can we make allergen detection methods more sensitive?

Poms RE, Capelletti C, Anklam E. Mol Nutr Food Res. (2004) 48(6):459-64

Processing also reduces extractability of proteins from foods like peanuts

Roasting dramatically alters the profile of peanut proteins

Roasted peanuts are used because they are the most widely consumed form and are considered to be the most allergenic.

Sayers et al unpublished.

Cupin Superfamily – 7S and 11S seed storage globulins

•The 11S and 7S seed storage globulins are large mulitmeric proteins with a cupin β-barrel motif

•They include allergens like peanut Ara h 1, Ara h 3,4 and soybean Gly m 5 (β-conglycinin )

0.0001

0.001

0.01

0.1

1

10

100***

******

******

***

A B C D

N-Ara h 1 batches

R-Ara h 1

IC50 (

µg

/ml)

0.0001

0.001

0.01

0.1

1

10

100***

******

******

***

A B C D

N-Ara h 1 batches

R-Ara h 1

IC50 (

µg

/ml)

Ara h 1 aggregates formed after boiling have lower allergenic activity than roasted Ara h 1

Blanc et al Mol Nutr Food Res 2011 55(12):1887-94

Native and Roasted Ara h 1 gave and IC50 of ~31-37ng/ml Boiled Ara h 1 had and IC50 of gave one of ~700-1200ng/ml

Unheated Boiled (15 min 100°C)

1mm

Roasted

1mm 1mm

At high concentration Ara h 1 forms an insoluble gel

A big issue with studying processed foods is protein insolubility •Is it IgE-reactive? •Is only soluble protein problematic?

4min:~1-2µm spherical structures

10min:~100µm clusters making sample turbid

15 min: continuous firm transparent gel

Rigby, Macierzanka, Mackie, Mills un published

• The disulphide-bonded , rigid structure of the prolamin superfamily confers resistance to thermal processing •This structure also makes the proteins stable to digestion •These properties may explain the potency of these allergens even though they are less abundant in peanut • The scaffold confers similar stability properties on lipid transfer proteins (LTPs), another potent type of food allergen like peanut Ara h 2/6

Prolamin Superfamily – 2S albumins

Ara h 2/6 unfolds when heated in water

•Ara h2/6 has to be heated to at least 110°C to be “denatured” but remains as a soluble mixture of monomers and dimers. •These properties may explain reported differences in the allergenicity of boiled compared to roasted peanuts

0

20

40

60

80

100

120

140

20 40 60 80 100 120

Time (minutes)

Ab

so

rba

nc

e (

28

0n

m)

unheated

heated

heated + glucose

molecular weight

markers

Native

Heated (15 min 110˚C)

Johnson et al (2010) Mol Nutr Food Res. 54(12):1701-10

Ara h 2/6 with an altered shapes is not recognised so well by human IgE ……………………..

N-Ara h 2/6 H-Ara h 2/6 G-Ara h 2/6 R-Ara h 2/60

50

100

150

200

p<0.0001

p=0.0002

p=0.003

IC50 (

ng

/mL

)

Ara h2/6 heated to 110°C looses its IgE binding capacity

IgE binds to native Ara h2/6 from roasted peanuts as well as raw peanuts

Vissers, et al PlosOne 2011, 6(8):e23998

Synapt G2-S HDMS spectrometer with nanoAcquity® UPLC and data analysis performed using Progenesis QI.

DIA-IM-MS provides a means of profiling peanut allergens Normalised quantity

distributions show, as expected the storage proteins allergens Ara h 3 and Ara h 1 comprise the most abundant proteins

0 200 400 600 Protein Index

0 200 400 600

Protein Index

Raw peanut

Roasted defatted peanut flour

Ara h 3 Ara h 1

Ara h 7

Ara h 10, 11

Ara h 8

Ara h 9

Ara h 6 Ara h 2

• Ara h 2,6 and 7 were the next most abundant

• Oleosin allergens Ara h 10 and 11 were less abundant in defatted peanut flour

• Ara h 8 and 9 were not abundant

• No evidence for the presence of the profilin allergen Ara h 5 could be found

Johnson, Sayers, Gethings, Balasundaram, Marsh, Langridge, Mills et al Anal Chem 2016, 88, (11), 5689-95.

Analysis of relative abundance by allergen type shows processing reduced abundance of cupin allergens

• Mechanically-blanched peanuts had lower levels of storage proteins in extracts

• Roasting reduced the relative abundance of storage protein allergens Ara h 1 and 3 relative to the 2S albumin allergens Ara h 2,6 and 7

• This is likely due to reduced extractability in the Tris-DTT-Rapigest buffer used and is consistent with the properties of the allergens

Johnson, Sayers, Gethings, Balasundaram, Marsh, Langridge, Mills et al Anal Chem 2016, 88, (11), 5689-95.

Effective extraction of peanut proteins requires reducing agent and detergents

• PBS was a poor extractant

• Tris-HCl was only able to extract protein effectively from raw peanut flour

• CHAPS -urea-DTT buffer was the most effective

• Tris-HCl-DTT- acid labile detergent (Rapigest) was intermediate but extracted a representative profile of peanut proteins by SDS-PAGE

B u f f e r 1 B u f f e r 2 B u f f e r 3 B u f f e r 4 B u f f e r 5

0 .0

0 .5

1 .0

1 .5

2 .0

mg

ex

tr

ac

te

d p

ro

te

in p

er

mg

to

ta

l p

ea

nu

t p

ro

te

in

PBS

Tris-HCl pH 8.8

Tris-HCl pH 8.8, DTT

Tris-HCl pH 8.8, DTT, Rapigest™

Wheel-mixing, 2h Sonication, 15 min, 60°C

CHAPS, urea-thiourea, DTT

Sayers et al Analyst 2016, 141, (13), 4130-41

Peanut

Buffe

r 2.1

Buffe

r 2.2

Buffe

r 2.3

Buffe

r 2.4

Buffe

r 2.5

0.0

0.2

0.4

0.6

0.8

1.0

mg

extr

acte

d p

rote

in p

er

mg

to

tal

pean

ut

pro

tein

Hazelnut

Buff

er 2

.1

Buff

er 2

.2

Buff

er 2

.3

Buff

er 2

.4

Buff

er 2

.5

0.0

0.5

1.0

1.5

2.0

2.5

mg

extr

acte

d p

rote

in p

er

mg

to

tal

haze

lnu

t p

rote

in

Peanut

Hazelnut

50 mM Tris-HCl pH 8.8

Urea DTT NaCl

DTT Rapigest

50 mM Tris-HCl pH 8.8

Urea DTT NaCl

DTT Rapigest


Buff

er 2

.1

Buff

er 2

.2

Buff

er 2

.3

Buff

er 2

.4

Buff

er 2

.5

0.00

0.05

0.10

0.15

Walnut

mg

ex

trac

ted

pro

tein

per

mg

to

tal

wa

lnu

t p

rote

in

Walnut 50 mM Tris-HCl pH 8.8

Urea

DTT NaCl

DTT Rapigest



• Optimal extraction was achieved for peanut and hazelnut ingredients using CHAPS, urea-thiourea, DTT with sonication 15mins at 60°C

• Walnut extraction is problematic, possibly due to polyphenols

Baricevic-Jones, Schäffer and Mills, unpublished

Even using chaotropes may not extract protein from some foods effectively

Improving allergen detection by optimising extraction – some issues!

Chaotropes and detergents may help extraction but …… • Adversely affect ELISAs (affecting plate coating

and denaturing antibodies) [although very sensitive assays may work because of sample dilution]

• Cannot be used in conjunction with LC -MS

How can we develop a targeted MS method as sensitive as an ELISA?

Skyline - Transition selection

Confirmatory MS/MS

Size screen (5-20 aa length)

Compositional screen (avoid Methionine)

BLAST - Check peptides are unique

MCPRED – prediction of missed cleavages

CONSeQuence – prediction of detection

Identification of non-redundant sequences in target protein(s)

Selection of peptides for targeted analysis

The curated peanut allergen sequence set was used to identify peptide targets generated by trypsin digestion.

Candidate peptide targets were identified using a bioinformatics approach and confirmed using discovery data sets obtained using DDA

(Orbitrap MS/MS). Sayers et al Analyst 2016, 141, (13), 4130-41

Protein Family Allergen UniProt ID

Subunit Mr (kDa)

Peptide Target Residues

Peptide Target Sequence

Peptide target name

Cupins

7S vicillin- globulin

Arah1 P43237 61.72

329-342 VLLEENAGGEQEER Arah1(P43237)329-342

555-577 DLAFPGSGEQVEK Arah1(P43237)555-577

11S legumin-globulin

Ara h 3 Q647H4 59.64

25-41 QQPEENACQFQR Arah3(Q647H4)25-41

372-384 SPDIYNPQAGSLK Arah3(Q647H4)372-384

Prolamins 2S Albumins

Ara h 2 Q6PSU2 17.99

103-115 CCNELNEFENNQR Ara h2(Q6PSU2)103-115

147-155 NLPQQCGLR Ara h2(Q6PSU2)147-155

Ara h 6 Q647G9 14.85 136-144 CDLDVSGGR Arah6(Q647G9)136-144

Ara h 7 B4X1D4 17.38 143-151 NLPQNCGFR Arah7(B4X1D4)143-151

Candidate peptides for targeted analysis

Sayers et al Analyst. 2016 ;141(13):4130-41.

Effect of thermal processing on detection of allergen peptide targets in MRM experiments

Ara h 1

Ara h 2, 6, 7

Ara h 3 • Peptide targets

in the cupin allergens were more prone to processing-induced effects

• Targets flanked by arginine residues showed greater thermostability.

VLLEENAGGEQEER

DLAFPGSGEQVEK

QQPEENACQFQR SPDIYNPQAGSLK (all)

CCNELNEFENNQR NLPQQCGLR

CDLDVSGGR

NLPQNCGFR


Roasted peanut flour incurred into cookie, chocolate dessert and chocolate bar matrices at 0, 3, 10, 50 mg protein/Kg

Peanut incurred matrices Extracted using Tris-HCl pH 8.8, DTT, Rapigest™ with sonnication at 60°C for 16 min

Effective extraction of peanut proteins requires reducing agent and detergents

• Tris-HCl-DTT- acid

labile detergent (Rapigest™) was used to prepare extracts of incurred matrices

• Extracts were then reduced, alkylated and digested

B u f f e r 1 B u f f e r 2 B u f f e r 3 B u f f e r 4 B u f f e r 5

0 .0

0 .5

1 .0

1 .5

2 .0

mg

ex

tr

ac

te

d p

ro

te

in p

er

mg

to

ta

l p

ea

nu

t p

ro

te

in

PBS

Tris-HCl pH 8.8

Tris-HCl pH 8.8, DTT

Tris-HCl pH 8.8, DTT, Rapigest™

Wheel-mixing, 2h Sonication, 15 min, 60°C


Sayers et al Analyst 2016, 141, (13), 4130-41

Protein Family Allergen UniProt ID

Subunit Mr (kDa)

Peptide Target Residues

Peptide Target Sequence

Peptide target name

Cupins

7S vicillin- globulin

Arah1 P43237 61.72

329-342 VLLEENAGGEQEER Arah1(P43237)329-342

555-577 DLAFPGSGEQVEK Arah1(P43237)555-577

11S legumin-globulin

Ara h 3 Q647H4 59.64

25-41 QQPEENACQFQR Arah3(Q647H4)25-41

372-384 SPDIYNPQAGSLK Arah3(Q647H4)372-384

Prolamins 2S Albumins

Ara h 2 Q6PSU2 17.99

103-115 CCNELNEFENNQR Ara h2(Q6PSU2)103-115

147-155 NLPQQCGLR Ara h2(Q6PSU2)147-155

Ara h 6 Q647G9 14.85 136-144 CDLDVSGGR Arah6(Q647G9)136-144

Ara h 7 B4X1D4 17.38 143-151 NLPQNCGFR Arah7(B4X1D4)143-151

Candidate peptides for targeted analysis


0 1 2 3 4 5 6

0

1

2

3

4

5

6

7

8

a m o le s o n c o lu m n ( lo g 1 0 )

Pe

ak

are

a (

log

10

)

b u ffe r

c h o c d e s s e rt

c h o c b a r

c o o k ie

0.9879R2 =

Arah2(Q6PSU2)147-155 peptide (NLPQQCGLR) SID on UPLC

• Retention time of 3.2-3.4 min

• Only the top four peptide concentrations have all 3 transitions

• Ratios between heavy and light peptides are variable with only often only single transition detected

• Significant matrix interference from chocolate bar

Buffer

Chocolate dessert

Cookie

Chocolate bar

Sayers et al (J Proteome Res in press)

0 1 2 3 4 5 6

2

3

4

5

6

7

8

9

1 0

a m o le s o n c o lu m n ( lo g 1 0 )

Pe

ak

are

a (

log

10

) R2 = 0 .9 9

b u ffe r

c h o c d e s s e rt

c h o c b a r

c o o k ie

NLPQQCGLR on ion key has improved sensitivity and reproducibility

Buffer Chocolate dessert

Cookie Chocolate bar

• Retention time of 13.8-14.2 min

• All 3 transitions observed across the concentration range

• Ratios between heavy and light peptides only variable at the lowest concentrations

• Matrix interference not observed except for chocolate bar and only at lowest concentrations


Targeted analysis of peanut in chocolate dessert – LoD and LoQ for best performing peptides

UPLC

• Ara h 1

– LOD = 1077.4 amoles on column

– LOQ = 3232.2 amoles on column

• Ara h 3



• Ara h 2



Ion Key

• Ara h 1



• Ara h 3



• Ara h 2



• Peptides love to fly in the ion key1! • LoD and LoQ values increased for all peptides • Best performing peptides have potential to provide

sensitivity required for allergen analysis


Profiling of peanut allergens in peanut flour standard using MRMs to support conversion from peptide to protein

Allergen molecule

•Targeted analysis returns lower than expected levels of Ara h 1 than expected. •This may relate to processing-induced reduction in solubility of Ara h 1 •Ara h 3 is over-estimated reflecting issues with peptide standard stability (deamidation?)

The analysis of peanut flour allowed development of conversion factors to go from peptide to peanut protein


Pilot analysis of peanut incurred in two matrices using a simple extraction procedure

• Arah2(Q6PSU2)147-155 could be quantified at the 10 ppm level in the chocolate dessert matrix also with three transitions;

• Conversion to peanut protein gave recoveries of 30-40% reflecting solubilisation of peanut in the extraction buffer;

• Performance similar to ELISA Sayers et al (J Proteome Res in press)

Mass spectrometry methods can have sufficient sensitivity........

BUT......there is much required to ensure future methods are robust! • Lack of sequenced genomes makes development of MS methods

for food allergens more difficult • Sample extraction and preparation (especially digestion) needs

further optimisation • Development of methods capable of dealing with processing-

induced modifications and diverse food matrices • Lack of reference materials and agreed ways of calculating and

reporting allergen which is meaningful for everyone – including patients! [THESE NEED TO BE IN PROTEIN TO BE USED IN RISK ASSESSMENT]

How much is too much?

[informing what to measure and how low do we

need to go]

Measuring how much [making sure we are measuring

what is important and at a relevant level]

• Helping to inform industry when to use PAL

• Helping patients to understand what PAL means for them

Manchester University: Rebekah Sayers, Phil Johnson, Justin Marsh, Anuradha Balasundaram, Aida Semic-Jusafagic, Angela Simpson, Adnan Custovic, Marina Themis, Ivona Baricevic-Jones, Huan Rao, Daniel Schäffer, Angela Simpson, Phil Couch, Iain Buchan, Chris Munro, Bushra Javed, Hadeer Mattar, Matt Sperrin

iFAAM collaborators: Sabine Baumgartner, Kathrin Lauter, Gavin O’Conner, Chiara Nitride, Karine Adel Patient, Hervé Bernard, Barbara Ballmer-Weber, Montserrat Fernandez-Rivas, Kirsten Beyer, Paul Turner, Audrey DunnGalvin, Jonathan Hourihane

The Team

Chemical Food Safety and Integrity

Units include : • Food chemical safety risk assessment • Methods for chemical food safety

analysis • Sampling, data quality assurance and

data analysis • Food fraud and authenticity • Chemical contaminants in food • Food allergens, additives and functional

ingredients

www.manchester.ac.uk/study/masters/courses/list/11707/msc-chemical-food-safety-and-integrity

Professor E N Clare Mills, Chair in Molecular Allergology, The University of Manchester. • Biochemist with research interests in food allergy and expertise in risk assessment and analysis including

bioanalysis and mass spectrometry.

• Led the IFAAM and EuroPrevall EU projects on food allergy and is a member of UK ACNFP and EFSA

GMO panel self-task allergenicity working group

René Crevel, Science Leader, Safety and Environmental Assurance Centre, Unilever • Responsible for advice and guidance on food allergy, risk assessment and management to and leading the

food allergy research programme.

• Chair, Food Allergy Task Force, ILSI-Europe.

• Member, UK's Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment.

Dr Martin D Rose FRSC, Independent professional services in food and environmental chemical

safety and Honorary Senior Lecturer at The University of Manchester

• Chartered chemist with > 31 years' experience in analytical chemistry and risk assessment relating to Food

Quality and Safety.

Former Head of the UK National Reference Laboratory and Fera Science lead for environmental

contaminants.

• Member of the EFSA CONTAM Panel and formerly on the EFSA ANS Panel.

Dr Mike Bromley, Senior Lecturer in Medical Mycology, The University of Manchester

• Founder of food analysis and diagnostic development company Genon Laboratories, providing analytical

diagnostics to detect all legislated allergens and GM material for the production of allergen- and GM-free

foods.

• Recently developed Next Generation Sequencing technologies to enhance food security with support from

the Technology Strategy Board and the FSA.

MSc Food Chemical Safety and Integrity Course Tutors

Sara Stead, Senior Strategic Collaborations Manager, Food and Environmental division, Waters

Corporation. • Strategic market development with a number of research applications in the Food and Environmental

sector

• Specialist in developing analysis for chemical residue and natural contaminants.

Dr Chiara Nitride, Lecturer in Proteomics of Food Allergy, The University of Manchester • Joined the University of Manchester in April 2017 from the EC Joint Research Centre in Geel developing

and validating quantitative food allergens methodologies

• With a background in food science and technology research is focussed on hazelnut and other tree nut

allergens.

manchester institute of biotechnology food allergen analysis: … · 2017-11-14 · victoria lee 1,...

Documents