Allergens are a major food safety concern and incidences of food allergy in industrialised populations has increased in recent times. One of the most common food allergies is that of peanuts. Food regulations for allergens exist in many countries and are being modified regularly as more is understood about allergens and the reactions they cause. This presentation describes the use of time-of-flight mass spectrometry to locate, identify and quantify an allergenic protein in both raw and roasted peanuts. Typical food processing (e.g. food processing) can alter the markers peptides present and amount that they are present in the samples which adds complexity to the analysis.

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The Analysis Of Allergens in Raw and
Roasted Peanuts using nanoscale UPLC &
Time-of-Flight Mass Spectrometry

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Presentation Overview
 Allergens Background
 Technologies for Allergen Analysis
 Experimental Workflow
– Sample Prep
– Instrument set-up
– Software
 Results
 Conclusions

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Background: Allergens
Overview
 Incidence of food allergy in industrialised populations is
increasing
 Effects for suffers can be fatal
 Food Regulations WW
– Reduce cross-contamination in factory
– Food Labelling

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Background: Allergens
Type of Food Categories

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Analysis of Allergens
Technologies
ELISA
PCR
MS
IncreasingCurrentUsage
IncreasingInstrument
Complexity&Price

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Workflow
SAMPLE PREPARATION
(1) Protein extraction (2) Tryptic digest
DATA ACQUISITION
Acquire data-independent MSE Data
SOFTWARE PROCESSING
PLGS
INSTRUMENT SET-UP
(1) nanoACQUITY UPLC ® (2) XevoTM QTof MS

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Workflow (1)
SAMPLE PREPARATION
 PART 1:
— Ara h1 protein extraction from raw and roasted peanut
 PART 2:
— Tryptic digest of raw and roasted Ara h1 extract (RapiGest™ SF)
 (PART 3:)
— Ara h1 identification and quantification in matrix (additional use
of ADH)

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nanoACQUITY UPLC
 Column:
– nanoACQUITY™ BEH C18, 75 mm x
150 mm
 Flow Rate:
– 300 nL/min
 Mobile Phase:
– A: 0.1% FA in Water
– B: 0.1% FA in Acetonitrile
 Gradient:
Workflow (2)
INSTRUMENT SET-UP
(1) nanoACQUITY UPLC ® (2) XevoTM QTof MS

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nanoACQUITY data
Raw peanut sample

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Xevo QTof MS
 Ionisation Mode:
– Electrospray Positive Ion
 Capillary:
– 3.3 V
 Cone:
– 25 V
 Source Temperature:
– 100oC
 LC/MSE Conditions:
– MS scan (Low CE): 6 V
– MSE scan (High CE ): 15 – 40 V
– Scan time: 0.6 sec
Workflow (2)
INSTRUMENT SET-UP
(1) nanoACQUITY UPLC ® (2) XevoTM QTof MS

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 UPLC-MSE provides ‘all the data, all the time’
– More information from a single analysis
 UPLC-MSE employs a simple methodology which
– Uses generic methods of acquisition
– Uses relevant Application Manager to mine data set
– ‘Acquire your data, Ask questions later!’
Workflow
DATA ACQUISITION
Acquire data-independent MSE Data

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Advantages of MSE with PLGS
Time-aligned spectra
Low energy fragmentation
High energy fragmentation
*

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Workflow
SOFTWARE PROCESSING
PLGS (ProteinLynx Global SERVER™ )
Databank: SwissProt
False Positive Rate: 4%
Fixed modification: Carbamidomethyl C
Variable modification: Acetyl N-Term,
Deamidation N, Deamidation Q, Met-
Oxidation, Hydroxy P
N-Linked Glycosylation
~10 ppm window for precursor ions;
~25 ppm window for fragment ions

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ProteinLynx Global SERVER™
Parameters
•Data preparation
Default parameters were used. Lock mass correction: 785.8426.
•Work flow
Databank: SwissProt
False Positive Rate: 4%
Fixed modification: Carbamidomethyl C
Variable modification: Acetyl N-Term,
Deamidation N, Deamidation Q, Met-
Oxidation, Hydroxy P
N-Linked Glycosylation
~10 ppm window for precursor ions;
~25 ppm window for fragment ions

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Peptide Sequence Identification:
GSEEDITNPINLR

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Peptide sequences observed in
both raw & roasted samples

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Relative intensities of peptide
sequences present in both samples

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Peptide Coverage
Raw and Roasted Samples
Signal peptide: 1-25
Raw Sample Roasted Sample

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Quantification of Ara H1
in Matrix

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Experimental Overview
 Aim:
– To identify and quantify Ara H1 in complex
matrix
 Samples:
 Two samples investigated – different
concentrations:
– Sample A – 1 : 3 (v/v) Sample 1 vs Standard
solution
– Sample B – 1 : 200 (v/v) Sample 1 vs Standard
solution

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Workflow
SAMPLE PREPARATION
 PART 1:
— Ara h1 protein extraction from raw and roasted peanut
 PART 2:
— Tryptic digest of raw and roasted Ara h1 extract (RapiGest™ SF)
 (PART 3:)
— Ara h1 identification and quantification in matrix (additional use
of ADH)

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Sample A – 1 : 3 dilution
MS – Low collision
energy data
MSE – High collision
energy data

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Sample B – 1 : 200 dilution
MS – Low collision
energy data
MSE – High collision
energy data

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Sample B – 1 : 200 dilution
Peptide Coverage

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Summary
 Challenges for analysis of allergenic proteins – many different
approaches already used
– Complexity added – Typical food processing (e.g. food processing)
can alter the markers peptides present / amount that they are
present in the samples
 Analytical tools and software can support confidence in the
results obtained
– ProteinLynx Global Server (PLGS) with intelligent filtering and
scoring routines minimizes occurrence of false positive results
– UPLC-MSE fragment ion exact mass data provides greater
confidence in protein identification in food samples