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ACI 150301

REVIEW

CURRENTOPINION Proteomic applications in food allergy: food

allergenomics

1528-4050 Copyright � 2015 Wolte

a b c d

Francesco Di Girolamo , Maurizio Muraca , Oscar Mazzina , Isabella Lante ,and Lamia Dahdahc

Purpose of review

To familiarize the reader with the recent developments in the identification of food protein allergens byproteomics mass spectrometry-based methods, named allergenomics.

Recent findings

The proteomic analysis of food protein allergens has became a hot topic in the food safety field in recentyears. Indeed, food allergies represent a current and relevant problem in clinical medicine. Several foodallergenomics studies have recently been performed, aiming at better understanding the cause ofsensitization to cow’s milk in breastfed infants and at assessing both the safety of food (e.g. transgenic)and in particular the allergenic properties of processed fish and seafood.

Summary

Food protein allergen characterization and quantification, together with the immunoglobulin E epitopemapping, will contribute to the diagnosis/prognosis of food allergy and will lead to a better safetyassessment of foods (e.g. novel transgenic foods).

Keywords

allergenomics, food allergens, mass spectrometry, proteomics

aDepartment of Laboratory Medicine, BambinoGesu Children’s Hospital,IRCCS, Piazza Sant’Onofrio, Rome, bDepartment of Women’s andChildren’s Health University of Padova, Via Giustiniani, Padova, cAllergyAllergy Unit, Bambino Gesu Children’s Hospital, IRCCS, Piazza San-t’Onofrio, Rome and dDepartment of Laboratory Medicine, San CamilloHospital, Viale Vittorio Veneto, Treviso, Italy

Correspondence to Francesco Di Girolamo, Department of LaboratoryMedicine, Bambino Gesu Children’s Hospital, IRCCS, Piazza Sant’O-nofrio 4, 00165 Rome, Italy. Tel: +39 06 68592176; fax: +39 0668592218; e-mail: francesco.digirolamo@opbg.net

Curr Opin Allergy Clin Immunol 2015, 15:000–000

DOI:10.1097/ACI.0000000000000160

INTRODUCTION

Immunoglobulin E (IgE)-mediated food-allergicreactions are emerging as an epidemic, and largelycontribute to the increased incidence of allergicdiseases in infants and preschool children [1]. Theyare estimated to affect between 1 and 10% of theworld’s population [2

&

]. Although several food aller-gies cause mild reactions, in some cases, they canevolve to severe clinical manifestations such as ana-phylaxis. Foods potentially triggering anaphylaxisreactions include milk, egg, soy, wheat, peanut, treenuts, fish, and shellfish. Milk, egg, and soy allergiesare more common in children, whereas fresh fruit,nut, and seafood allergies are common in adults [2

&

].Clearly, preventing the use of the offending food isa key measure in the management of the disease.With the industrialization progress, the effective-ness of this approach is shrinking due to theincreased complexity of food composition, ina-dequate food labelling, and increasing contamina-tion of some allergens in the diet.

To this aim, the identification, characterization,and quantification of all food protein allergens(FPAs) and their post-translational modifications(PTMs), as well as the detection of the molecular

rs Kluwer Health, Inc. All rights rese

determinants that make a protein behave as anallergen, are highly desirable.

Strategies used to detect and quantify FPAs haveevolved over the past years. In the past decade, theadvent of modern mass spectrometry technologiesand the improvement of bioinformatic tools andgenomic data banks widened our horizons, creatingthe ‘new’ science of proteomics. This term encom-passes the large-scale experimental analysis ofproteins, introduced in a wide range of medicalapplications, from cancer medicine [3] to cardiology[4] and allergy [5,6]. The potentials of proteomicapproaches in food allergies are so large that we are

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ACI 150301

KEY POINTS

� Allergenomic applications have revolutionized the foodallergy field, providing innovative information andaccelerating the analysis of food protein allergens.

� The food allergenomic studies have recently beenperformed to better understand the cause ofsensitization to cow’s milk in breastfed infants and toassess both the safety of food (e.g. transgenic) and theallergenic properties of processed fish and seafood.

� Food protein allergen characterization andquantification, together with the IgE epitope mapping,will contribute to the diagnosis/prognosis offood allergy.

Food allergy

now at the dawn of the era of food allergenomics.The mass spectrometry-based proteomic analyses ofnovel FPA and the IgE–FPA epitope mapping areproviding the following essential information atvarious levels [7,8,9

&&

]:

(1)

2

understanding the mechanisms of the immunereactions;

(2)

developing novel and effective protocols forfood allergy diagnosis/prognosis;

(3)

providing information regarding patient’sclinical history;

(4)

offering a safety assessing of foods (e.g. noveltransgenic foods);

(5)

evaluating the PTMs occurring during techno-logical processes of food, in order to assess theallergenic properties of processed foodstuffs.

Here, we describe the evolution over the yearsof the methods used in identifying and quantify-ing FPA, focusing on the innovative food allerge-nomics and their applications within the past12 months.

FOOD PROTEIN ALLERGEN DETECTION BYCOMMONLY USED METHODS

Historically, the detection of FPAs has been per-formed using antibody-based assays, with or with-out protein-sequencing techniques. In these assays,the IgE are collected from allergic patients and FPAsare detected through their direct binding.

The most used technique in food allergy isIgE immunoblotting. It detects FPAs following afood protein separation by one-dimensional ortwo-dimensional gel electrophoresis (1-DE, 2-DE),and a subsequent protein transfer from the gel to amembrane (typically nitrocellulose or polyvinyli-dene difluoride). After the incubation of the

www.co-allergy.com

membrane with serum samples from allergicpatients, the specific IgE binds the FPA.

Such an analytical strategy allows visualizingpossible IgE–FPA immune reactions, assigning theFPA of interest on a reference electrophoretic map(Fig. 1a).

As an evolutionary step, to determine the N-terminal amino acid sequence of FPA, the Edmandegradation techniques were integrated (Fig. 1b)during the late 1990s. As described by Park et al.[10], this innovative technique allowed identifi-cation of the FPA via a sequence homology assign-ment.

Traditionally, the quantification of FPA is per-formed by enzyme-linked immunosorbent assay(ELISA), in which case specific IgG from immunizedanimals bind a target FPA coated onto microplatewells (Fig. 1c) [11].

The above-described immunological methodsallowed FPA visualization, sequencing, and quanti-fication, but had a series of limitations. They weredependent on the complexity of the food matrix,the presence of allergenic contaminants derivedfrom other foods, and the variable specificity ofthe antibody preparations of the commercial ELISAkit. Moreover, the absence of animal and plantgenome databases prevented the possibility of mol-ecular studies for FPA characterization together withthe IgE epitope mapping.

FOOD ALLERGENOMICS

Proteomic mass spectrometry-based methods over-come these obstacles, allowing unambiguous detec-tion and quantification of traces of single ormultiple FPAs and their isoforms even in processedproducts, where physical or chemical alterationsmight affect the protein stability preventing itsidentification by the above-described methods.

The precursors of food allergenomic applicationintegrated matrix-assisted laser desorption ioniz-ation–time of flight (MALDI-TOF) mass spectrometrywith the Edman degradation technique for a bettercharacterization of the apple and peach FPAs [12].They purified both fruit proteins by reverse-phasehigh-performance liquid chromatography (HPLC)and separated each fraction by 1-DE gel. The lipid-transfer proteins were identified as FPAs by specificIgE immunodetection and immunoblot inhibitionassays with sera from allergic patients, and werecharacterized by the N-terminal amino acid sequenc-ing and linear MALDI-TOF mass spectrometryanalysis.

With the advent of high-resolution mass spec-trometry technologies and the improvement of bio-informatic tools and genomic data banks, food

Volume 15 � Number 00 � Month 2015

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ACI 150301

FOOD PROTEIN EXTRACTION

1-DE 2-DE

IMMUNOBLOTTING WITH SERA OF AP

(a)

FOOD PROTEIN EXTRACTION

1-DE 2-DE

IMMUNOBLOTTING WITH SERA OF AP

(b)

N-TERMINAL AA SEQUENCEBY EDMAN DEGRADATION

FPA SEQUENCING

FPA TARGET

FPA QUANTIFICATION

ELISA

IgG

(c)

FPA VISUALIZATION

FIGURE 1. Schematic representation of the process of food protein allergen (FPA) discovery and quantification usingconventional biochemical methods. Two-dimensional gel electrophoresis (2-DE) purification is followed by a reaction withserum from allergic patients (AP) for FPA visualization (panel a) and N-terminal amino acid sequence by Edman degradationtechnique for FPA sequencing (panel b). The scheme for quantification of FPA target in food extracts using ELISA test is shownin panel c. 1-DE, one-dimensional gel electrophoresis; ELISA, enzyme-linked immunosorbent assay.

Proteomic applications in food allergy Di Girolamo et al.

allergenomics became a powerful tool for compre-hensive FPA characterization and quantification. Inthis regard, the first application of modern allerge-nomic strategy for the study of FPA using massspectrometry data has been applied during the2000 [13]. They separated wheat flour proteins bythe 2-DE approach and characterized the specificFPAs by immunoblot analysis, with sera of 10 aller-gic patients with Baker’s asthma, and MALDI-TOFmass spectrometry analysis.

FOOD PROTEIN ALLERGENCHARACTERIZATION BY ALLERGENOMICAPPROACHDue to the low amounts of several FPAs in foodsamples, their characterization often needs a con-centration step. Protein extraction and precipitationrepresent the most common pre-analyticalapproaches able to remove undesired materialsand to concentrate traces of proteins, but the mostused pre-treatment methodology in the food pro-teomic field is represented by the combinatorialpeptide ligand library (CPLL) technique [14–17].

To date, the qualitative gel-based proteomicmethod represents the standard approach in the foodallergenomic field to identify and characterize anovel FPA.

1528-4050 Copyright � 2015 Wolters Kluwer Health, Inc. All rights rese

As shown in Fig. 2a, it consists of separating theprotein extract by the 2-DE approach and a sub-sequent staining with regular protein dye (e.g. col-loidal Coomassie blue). After taking a picture, thegel is blotted against the serum of allergic patients,and the positive IgE–FPA spots are compared to thegel plate and then excised. Each of them is separ-ately hydrolysed using specific enzymes (e.g. tryp-sin), and the resulting peptides are separated byliquid chromatography systems and analysed bymass spectrometry (MS)or tandem mass spec-trometry (MS/MS) platforms for FPA identification.

The mass spectrometry analyses are typically per-formed by a MALDI-TOF mass spectrometry instru-ment. It allows protein identification by peptide massfingerprinting (PMF), by which the molecular massesare compared to the theoretically expected trypticpeptide masses for each entry into the database.The MS/MS analyses are carried out by MALDI-TOF/TOF or electrospray ionization-MS/MS (ESI-MS/MS)platforms. Data provided from these instrumentsdetermine structural information on the peptide’ssequence, rather than only their mass, increasingthe specificity and the discrimination of the search.

The informatic tools represent decisive supportfor the qualitative allergenomic analyses, for theinterpretation of the great amount of MS and MS/

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ACI 150301

SAMPLE

PRE-TREATMENT

2-DE

IMMUNOBLOTTING

WITH SERA OF AP

(a)

FPA

CHARACTERIZATION

AND

IgE EPITOPE MAPPING

STAINING

ALLERGEN MAPPROTEIN MAP

PROTEIN

HYDROLISIS

LC-MS/MS

Label withfluorescent dye(wavelenght 3)

FoodSample 2

FoodSample 3

FoodSample 1

Label withfluorescent dye(wavelenght 1)

Label withfluorescent dye(wavelenght 2)

Three samples

are mixed and

analysed together

(b)

Relativequantification

of FPA

FIGURE 2. Schematic representation of the process of FPA discovery and quantification using allergenomic approaches. Theidentification of FPA is operated by LC-MS/MS of individual IgE-positive spots (panel a). Panels b and c show the relativequantification of FPA by 2D-DIGE and label-based techniques, respectively. A scheme for absolute quantification of FPA infood extracts using targeted proteomics is shown in panel d. 2D-DIGE, 2D-differential gel electrophoresis; FPA, food proteinallergen; IgE, immunoglobulin E; MS, mass spectrometry.

Food allergy

MS data, and for the FPA identification via highlyperformant, in-house, and on-line software programssuch as Mascot, OMSSA, and SEQUEST, in combi-nation with constantly updated public databases,particularly those managed by the National Centerfor Biotechnology Information (NCBI) and SwissProt[18].

A significant weakness of the qualitative aller-genomic studies is due to the still partial character-ization of animal and plant genomes and theincomplete annotation of gene functions.

Even if there are some gaps in the full descrip-tion of animal and plant proteomes, protein identi-fication is, however, possible – thanks to theapplication of homology searching (Basic LocalAlignment Search Tool searching) on the referenceproteome databases. The qualitative analysis of nov-el FPAs has became a hot topic in the food safetyfield in recent years.

4 www.co-allergy.com

Table 1 [19–31,32&

] reports some examples ofallergenomic contributions to clinical management.In cow’s milk allergy (CMA), some infants experi-ence symptoms caused by cow’s milk proteinsingested by their mother and passed throughhuman milk. They are sensitive to maternal milkelimination, but cow’s milk proteins have neverbeen convincingly found in breast milk [33].Recently, Orru et al. [19] identified intact bovinealpha-S1-casein in human colostrum using a pro-teomic approach for direct protein identification.On this basis, bovine alpha-1-casein was identifiedas a possible cause of sensitization to cow’s milk inexclusively breastfed predisposed infants.

Rosmilah et al. [20] characterized the major FPAsof long-tail tuna (Thunnus tonggol). In addition toparvalbumin, they characterized two other majorFPAs of T. tonggol, establishing that both thermo-stable and thermolabile proteins are important in

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ACI 150301

Proteinhydrolysis

Pool peptide samples

nano LC-MS/MS analysis

iTRAQ reagent114,115,116,117

Proteinhydrolysis

ICAT light ICAT heavy

Pool peptide samples

ICAT-tagged peptide purification and

nano LC-MS/MS analysis

1:2

FoodSample 2

FoodSample 1

FoodSample 1

FoodSample 2

FoodSample 3

FoodSample 4

Relativequantification

of FPA

Relative

quantification

of FPA

(c)

Proteinhydrolysis

FoodSample

Proteinextraction

Add standardpeptides

StandardPeptide

Absolutequantification

of FPAusing target peptides

(d)

Proteomic applications in food allergy Di Girolamo et al.

local tuna allergy and should be included in diag-nostic strategies. Tomm et al. [21] identified novelFPAs from Nile perch (Lates niloticus) and cod (Gadusmorhua), with the aim to improve avoidance strat-egies. Allergic reactions can result from not onlyingesting seafood but also breathing vapours duringtheir cooking. Indeed, occupational asthma is amajor risk among workers involved in the seafoodindustry. Traditional methods identified tropomyo-sin [34] as the potential allergen responsible forasthma in different seafood. Using the proteomicapproach described in Fig. 2a, Abdel Rahman et al.[22] identified also arginine kinase and sarcoplasmiccalcium-binding protein as significant FPAs ofnorthern shrimp.

Kamath et al. [23] analysed the impact of heatingon prawn FPAs and demonstrated that it increasedthe antibody reactivity to prawn FPAs, as well as tothe derived fragments.

Nitride et al. [24] characterized a new FPA ofhazelnut, providing a new platform to plan innova-tive diagnostic and therapeutic interventionapproaches to treat hazelnut allergy.

Hebling et al. [25] demonstrated that thermalprocessing of peanuts and their derivatives alterpeanut protein structures, preventing accuratedetection of FPAs by most immunochemical andtargeted screening tests.

Correct food labelling is currently the mosteffective means of protecting allergic patients. Inthis regard, Costa et al. [26] compared allergenomicapproaches with ELISA and real-time PCR (RT-PCR)

1528-4050 Copyright � 2015 Wolters Kluwer Health, Inc. All rights rese

techniques to evaluate their performance in thedetection of hazelnut FPAs in model chocolates.They demonstrated that all the three methods wereappropriate for the identification of hazelnut incomplex foods such as chocolates.

Krishnan and Chen [27] raise the possibility ofusing waxy mutants of rice (the microscopic struc-ture of rice is similar to the wax one) in the diet ofpatients sensitized to the 56-kDa rice FPA (granule-bound starch synthase).

Finally, Monaci et al. [28] focused on three FPAcategories including egg, milk, and soy that mightaccidentally contaminate cookies chosen as foodmodel. They used a micro-HPLC coupled to a dualcell linear ion trap mass spectrometry to develop arapid and sensitive multi-FPA detection method.

FOOD PROTEIN ALLERGENQUANTIFICATION BY ALLERGENOMICAPPROACHESAs several foods can cause severe allergic reactions,understanding the level of FPA in food is desirable. Inthis respect, currently there is an urgent need for theabsolute quantification of FPAs for food safety pur-poses.

Quantitative proteomic methods can be classi-fied as relative and absolute. The aim of relativequantification is to establish the differencesin protein expression between two (or more) pro-teomes. Absolute quantification, instead, aims atquantifying the amount of proteins/peptidespresent in the sample.

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ACI 150301

Tab

le1

.Fo

odal

lerg

enom

ics

from

acl

inic

alpo

intof

view

(wha

tca

nw

eex

pect

from

alle

rgen

omic

tech

niqu

es?)

Clin

ica

lu

seFo

od

Alle

rgen

om

icco

ntri

bu

tio

nTe

chni

cal

met

hod

Ref

eren

ce

Offe

ring

asa

fety

asse

ssin

gof

food

s:un

mas

khi

dden

alle

rgen

sin

smal

l,pr

evio

usly

unsu

spec

ted

amou

nts

Cow

’sm

ilkId

entif

icat

ion

ofin

tact

bovi

neal

pha-

S1-c

asei

nin

the

hum

anco

lost

rum

Term

and

pret

erm

hum

anco

lost

rum

sam

ples

wer

etre

ated

with

CPL

Lap

proa

chto

capt

ure

the

who

lepr

oteo

me.

DE

was

used

topr

otei

nse

para

tion

and

MS

topr

otei

nid

entif

icat

ion

Orr

uet

al.

[19]

Dev

elop

men

tofno

vela

ndef

fect

ive

pro-

toco

lsfo

rfo

odal

lerg

ies

diag

nosi

sLo

ng-ta

iltu

na(T

hunn

usto

nggol

)

Cha

ract

eriz

atio

nof

two

alle

rgen

icpr

otei

nsto

bein

clud

edin

diag

-no

stic

stra

tegie

s

Fish

prot

eins

wer

ese

para

ted

by1-D

Ean

d2-D

E.Ig

Eim

mun

oblo

tting

ofra

wex

tract

was

perfor

med

with

sera

from

fish-

alle

rgic

patie

nts.

Sele

cted

alle

rgen

icpr

otei

nsp

ots

wer

eex

cise

dan

dan

alys

edby

MS

Rosm

ilah

etal

.[2

0]

Nile

perc

h(L

ates

nilo

ticus

)an

dco

d(G

adus

mor

hua)

Cha

ract

eriz

atio

nne

wal

lerg

enic

prot

eins

apar

tfro

mpa

rval

bum

in2-D

Ew

asus

edto

sepa

rate

the

mus

cle

prot

eins

ofN

ilepe

rch

and

cod.

IgE

imm

unob

lotti

ngof

raw

extra

ctw

aspe

rfor

med

with

sera

from

fish-

alle

rgic

patie

nts.

IgE-

reac

tive

prot

eins

wer

ede

tect

edan

did

entif

ied

usin

gM

S

Tom

met

al.

[21]

Prov

idin

gin

form

atio

nre

gar

ding

patie

nt’s

clin

ical

hist

ory

Shrim

pA

rgin

ine

kina

sean

dsa

rcop

lasm

icca

lciu

m-b

indi

ngpr

otei

nch

arac

-te

rize

das

signi

fican

tFPA

byin

hala

tion

DE

was

used

tose

para

teth

esh

rim

ptis

sue

prot

eins

.Ig

Eim

mun

oblo

tting

ofra

wex

tract

was

perfor

med

with

sera

from

shrim

p-al

lerg

icpa

tient

s.Ig

E-re

activ

epr

otei

nsw

ere

dete

cted

and

iden

tifie

dus

ing

MS

Abd

elRa

hman

etal

.[2

2]

Ass

ess

the

alle

rgen

icpr

oper

ties

ofpr

o-ce

ssed

food

stuf

fsPr

awn

Hea

ting

can

incr

ease

the

alle

r-ge

nici

tyPr

otei

nsfrom

raw

and

heat

-pro

cess

edbl

ack

tiger

praw

nw

ere

sepa

rate

dby

1-D

E.Ig

Eim

mun

oblo

tting

ofra

wex

tract

was

perfor

med

with

sera

from

shel

lfish

alle

rgic

patie

nts.

IgE-

reac

tive

prot

eins

wer

ede

tect

edan

did

ent-

ified

usin

gM

S

Kam

ath

etal

.[2

3]

Dev

elop

men

tofno

vela

ndef

fect

ive

pro-

toco

lsfo

rfo

odal

lerg

ies

prog

nosi

sH

azel

nut

Iden

tific

atio

nof

FPA

and

prop

osal

ofa

new

plat

form

for

diag

nost

ican

dth

erap

eutic

inte

rven

tions

DE

was

used

tose

para

teth

eha

zeln

utpr

otei

ns.

IgE

imm

unob

lotti

ngof

raw

extra

ctw

aspe

rfor

med

with

sera

from

haze

lnut

-alle

rgic

patie

nts.

IgE-

reac

tive

prot

eins

wer

ede

tect

edan

did

entif

ied

usin

gM

S

Nitr

ide

etal

.[2

4]

Pean

utTh

erm

alpr

oces

sing

can

hide

prot

ein

stru

ctur

es,

prev

entin

gac

cura

tede

tect

ion

ofFP

A

Rela

tive

quan

tific

atio

nof

alle

rgen

sin

pean

utly

sate

sw

asac

com

plis

hed

byla

bel-f

ree

LC-M

S/M

Sm

etho

dolo

gie

s,by

whi

chpe

anut

alle

rgen

pept

ides

exhi

bitin

ga

diffe

ren-

tialM

Sre

spon

sein

raw

vers

usro

aste

dpe

anut

sw

ere

cons

ider

edto

beca

ndid

ate

targ

ets

ofth

erm

alm

odifi

-ca

tion

Heb

ling

etal

.[2

5]

Haz

elnu

tId

entif

icat

ion

inco

mpl

exfo

ods

such

asch

ocol

ates

ELIS

A,

LC-M

S/M

San

dre

al-ti

me

poly

mer

ase

chai

nre

ac-

tion,

wer

eev

alua

ted

for

thei

rpe

rfor

man

cere

gar

ding

the

dete

ctio

nof

haze

lnut

alle

rgen

sin

mod

elch

ocol

ates

Cos

taet

al.

[26]

Prod

uctio

nof

hypo

alle

rgen

icfo

ods

Rice

Prod

uctio

nof

56-k

Da

FPA

–free

stra

ins

DE

was

used

tose

para

teth

epr

otei

nsof

rice

.Ig

Eim

mun

oblo

tting

ofra

wex

tract

was

perfor

med

with

sera

from

mai

ze-a

llerg

icpa

tient

s.Ig

E-re

activ

epr

otei

nsw

ere

dete

cted

and

iden

tifie

dus

ing

MS

Krish

nan

and

Che

n[2

7]

Offe

ring

asa

fety

asse

ssin

gof

food

s:de

velo

ping

rapi

dde

tect

ion

met

hods

Egg,

milk

,an

dso

yId

entif

icat

ion

ofm

ultip

leFP

Ain

cook

ies

Mic

ro-H

PLC

coup

led

toa

dual

cell

linea

rio

ntra

pM

Sto

deve

lop

ara

pid

and

sens

itive

mul

ti-FP

Ade

tect

ion

met

hod

Mon

aciet

al.

[28]

Food allergy

6 www.co-allergy.com Volume 15 � Number 00 � Month 2015

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ACI 150301

Tab

le1

(Con

tinue

d)

Clin

ica

lu

seFo

od

Alle

rgen

om

icco

ntri

bu

tio

nTe

chni

cal

met

hod

Ref

eren

ce

Cow

’sm

ilk,

egg

inw

ine

Sim

ulta

neou

sde

term

inat

ion

and

quan

tific

atio

nof

trace

sM

S-ba

sed

appr

oach

was

used

for

sim

ulta

neou

sde

term

i-na

tion

and

quan

tific

atio

nof

trace

sof

case

inat

ean

deg

g-w

hite

pow

ders

inw

ine

Mon

aciet

al.

[29]

Alle

rgen

anal

ysis

info

ods

Whe

atQ

uant

itativ

ean

alys

isan

dFP

Apr

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Proteomic applications in food allergy Di Girolamo et al.

1528-4050 Copyright � 2015 Wolters Kluwer Health, Inc. All rights rese

Allergenomic approaches for relative proteinquantification are based on 2-DE, demonstratingmodulations in protein expression by comparisonof gels from different food samples. To reduce gel-to-gel variability, 2D-differential gel electrophoresis(2D-DIGE) was developed. This technique is ableto assess protein profile changes from two or threesample runs on a single 2D-gel [35] (Fig. 2b).

An alternative and very common method forFPA quantification is based on LC-MS/MS technol-ogy. Mass spectrometry-based methods for relativequantification of allergens are commonly per-formed by label-based approaches (Fig. 2c), whereasabsolute quantification is carried out by label-freestrategies (Fig. 2d).

Recently, an innovative label-free targeted pro-teomic strategy based on the shotgun procedure,called selected reaction monitoring (SRM), hasemerged [36]. Briefly, after extraction from foodmatrices, the protein molecules are enzymaticallydigested and subsequently analysed by LC-MS/MSapproaches using selected proteotypic peptides.

Selected reaction monitoring is now consideredthe ‘gold standard’ for absolute quantification ofproteins and their application is exponentiallyincreasing in the allergenomic field because it per-mits one-step monitoring of more than a single FPA.

Monaci et al. [29] used the mass spectrometry-based approach for the development of an effectivemethod for simultaneous determination and quanti-fication of traces of caseinate and egg-white powders– fining agents typically employed for wine clarifi-cation that could represent a potential risk for allergicpatients. The amount of FPA in wheat grain is stilllargely unknown. In this respect, Uvackova et al. [30]used the mass spectrometry-based method for quan-titative analysis and FPA profiling in wheat varieties.Data suggested that this mass spectrometry-basedmethodology is also suitable for other food matrices.

The development of feasible mass spectrometry-based methods for absolute quantification of FPA ina food matrix with results traceable to the inter-national system of units is a step towards significantcomparison of FPA data among laboratories. To thisaim, Cryar et al. [31] applied the mass spectrometry-based procedure for absolute quantification of lyso-zyme in wine as a model system.

Finally, Flodrova et al. [32&

] investigated therelative quantitative changes of cereal FPA after foodtechnological processing, namely wheat couscousproduction and barley malting. Their resultssuggested that relative mass spectrometry-basedallergenomics is a universal and sensitive methodfor fast and reliable monitoring of quantitativechanges during food processing. Such informationis clearly important for allergic patients.

rved. www.co-allergy.com 7

CE: Namrta; ACI/150301; Total nos of Pages: 8;

ACI 150301

Food allergy

CONCLUSION

The proteomic platforms, in particular, mass spec-trometry, are becoming decisive to the progress ofmodern structural immunology, contributing to theevaluation of FPA related to food allergies.

The recent technological advances of nano-LC-MS/MS systems, and computational tools for massspectrometry data analysis and the continuousupdate of public databases enabled a big step for-ward in the allergenomic studies.

Allergenomic applications provide innovativeinformation and accelerate the analysis of FPA,compared with the conventional methods basedonly on immunological techniques such as immu-noblot and ELISA. Currently, the application of thismethodology requires sophisticated technologicalplatforms and informatic infrastructures to analysethe large number of data sets produced, and requireshighly skilled personnel for its application, makingit too expensive for most routine analyses. How-ever, the costs involved are rapidly falling, thusfavouring a more widespread use of this method-ology.

Acknowledgements

None.

Financial support and sponsorship

The study was supported by San Camillo Hospital,Treviso, Italy Fellowship Program to M.M.

Conflicts of interest

There are no conflicts of interest.

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