This presentation describes development of a routine tandem quadrupole LC/MS/MS method for milk and egg allergens based on proteomic studies to identify the allergenic peptide markers. Initial studies are done using a proteomic workflow and quadrupole time of flight mass spectrometry.

1. ©2015 Waters Corporation 1
Analysis of Milk and Egg Allergens
in Wine Using UPLC-MS
Work performed in collaboration with:

2. ©2015 Waters Corporation 2
Presentation Overview
 Background
– Allergenic proteins in wine
– MS technology for allergen analysis
 Establishing a Routine Workflow on
Tandem Quad MS
– QTof to Tandem quad MS
– Software to Tandem quad MS
 Conclusions

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Milk and Egg Proteins for
Wine Clarification
 Clarification is an important step in wine making
– Remove phenolic compounds (e.g. tannins)
 Fining agents include egg whites (albumin) and
milk (casein)
– Forms insoluble complex that settles at the bottom
– Wine is filtered to remove these finings
– Minimal quantities are used to achieve the desired
result without stripping too much flavour
 Finings should be removed when the wine is
clarified.
 In some countries it is a legal requirement to
state any potential allergens on the label

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

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What information can MS detection
provide?
 Analyse peptide markers of the protein causing the
allergic reaction
 Targeted and specific m/z analysis
 Quantifiable technique
 Capability to modify / optimise routine methods for
challenging matrices (without additional cost)
 Potential to use a multi-allergen approach

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Strategy 1
Instrument-based strategy to identify egg
and milk peptide markers using
Xevo G2 QTof MS

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Bottom-up Proteomic Experiment
1. Enzyme
digestion
2. UPLC
separation
Precursor
ions
MSE product
ions
3. MS
analysis
4. Data
interpretation

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Food Proteomic Workflow
SAMPLE PREPARATION
(1) Tryptic digest (2) ADH addition
DATA ACQUISITION
Acquire data-independent MSE Data
SOFTWARE PROCESSING
PLGS & IdentityE and Proteomic database (e.g. UniProt)
ANALYTICAL SYSTEMS
(1) ACQUITY UPLC ® (2) XevoTM G2 QTof

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Food Proteomics Workflow
Software processing
High energy product ion data gives increased confidence in peptide sequence identification
Markers are from a SINGLE protein
Unique marker peptides sequences listed here

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Xevo G2 QTof advantages for
determining suitable peptide markers…
NNPFYFPSR
DLAFPGSGEQVEK
VLLEENAGGEQEER
ISMPVNTPGQFEDFFPASSR

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VERIFYE
Transferring research data to routine analysis
Exact mass data is translated into MRMs with VERIFYE

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VERIFYE
…Proteotypic Peptide Review …MRM Method Generation

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Strategy 2
Software-based strategy to identify peptide
markers

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Skyline Experimental Design
Peptide
settings
Transition
settings
MRM
generation

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Skyline versus VERIFYE
 Advantages
– Good solution for customers who have invested in tandem quad MS
for allergen analysis
 Disadvantages
– Skyline can provide 100s potential MRM transitions
– Need to work through the list to determine
• Specificity for the matrix
• Sensitivity of the transition
– VERIFYE uses the QTof data and so the list is from instrumental
data

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Milk and egg allergen in milk:
Sample prep development
 Samples:
– Red and white wine, fortified before/after extraction
 Tested extraction/concentration protocols:
1. Ultrafiltration (cut-off 3kDa and 10 kDa)
2. precipitation with acetone
3. precipitation with acetone/TCA
4. precipitation with KDS
5. precipitation with ethanol
 Protein pellet suspended in a solution of 120mM Tris 7M urea 2M
thiourea
 Best sample prep results:
– Precipitation with cold acetone or ethanol

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ACQUITY UPLC parameters
(ACQUITY UPLC parameters have not yet been fully optimised)
LC system ACQUITY UPLC I-Class
Column BEH130 C18 UPLC column
2.1 x 150mm
Flow rate 0.5ml/min
Column temp 40°C
Solvent A Water + 0,1% formic acid
Solvent B ACN 0,1% formic acid
Time(min) A B
Initial 85 15
1.00 85 15
11.00 30 70
12.00 30 70
12.50 0 100
14.00 0 100
15.50 85 15
17.00 85 15

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Milk allergens:
Targeted peptides and MRMs
Peptide
Precursor
(m/z)
Product
(m/z)
YLGYLEQLLR (casein S1) 423.2 529.3
634.4 658.4
634.4 771.5
634.4 934.5
VPQLEIVPNSAEER (casein S1) 527.6 802.4
790.9 779.5
790.9 802.4
790.9 1014.5
FFVAPFPEVFGK (casein S1) 692.9 465.2
692.9 676.4
692.9 920.5
692.9 991.5
ALNEINQFYQK (casein S2) 456.6 827.4
684.3 713.4
684.3 827.4
684.3 940.5
FALPQYLK (casein S2) 490.2 332.2
490.2 551.3
490.2 648.4
490.2 761.5

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Egg allergens:
Targeted peptides and MRMs
Peptide Precursor
(m/z)
Product
(m/z)
DILNQITKPNDVYSFSLASR (ovalbumin) 761.0 767.4
761.0 930.5
761.0 1355.7
1141.1 1355.7
GGLEPINFQTAADQAR (ovalbumin) 563.3 732.4
844.4 860.4
844.4 1007.5
844.4 1121.5
844.4 1331.7
ELINSWVESQTNGIIR (ovalbumin) 620.3 673.4
620.3 888.5
930.0 1017.5
930.0 1116.6
EVVGSAEAGVDAASVSEEFR (ovalbumin) 670.3 853.4
670.3 924.4
1005.0 1110.5
1005.0 1266.6

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Peptide Identification & Confirmation
Using ACQUITY UPLC & Xevo TQ-S
1. Retention time
2. Standard MRM transitions

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Peptide Identification & Confirmation
Using ACQUITY UPLC & Xevo TQ-S
1. Retention time
2. Standard MRM transitions
3. Standard MRM transitions & full scan data
4. Product ion scanning confirmation
(PICs)

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Method Development with Skyline
Use of RADAR for Food Matrices
- Parallel MRM with full scan data acquisition
}

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Method Development with Skyline
Use of RADAR for Food Matrices
 Once the most selective, and then the most sensitive MRMs have been selected for a subset of
food matrices, RADAR can be used to support routine analysis
CaseinS1-
YLG
CaseinS1-
FFV
Ovalbumin-
EVV
Ovalbumin-
GGL
Ovalbumin-
DILOvalbumin-
ELI
CaseinS2-
FAL
CaseinS1-
VPQ
CaseinS2-
ALN
4
3
21
6
5
Full scan
MRMs

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Peptide Protein Charge state:
m/z
GPFPIIV β-CN +1: 742.4490
FFVAPFPEVFGK α-S1-CN +2: 692.8695
HQGLPQEVLNENLLR α-S1-CN +2: 880.4770
YLGYLEQLLR α-S1-CN +2: 634.3568
Comparing RADAR data with
Journal Citations
L. Monaci, I. Losito, F. Palmisano, M. Godula & A. Visconti (2011): Food
Additives & Contaminants: Part A: Chemistry, Analysis, Control, Exposure & Risk Assessment,
28:10, 1304-1314
XIC of m/z
742.4
RT – 5.33 or
6.13

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Peptide Identification & Confirmation
Additional confirmation using PICs
 Food processing can affect peptide response observed and there
may be other similar proteins present in complex food products
– PEPTIDE SPECIFICITY is essential
PIC scan for peptide
DLAFPGSGEQVEK
y and b ion fragments(*)
*
* *
*
*
* *
*
*

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Peptide Identification & Confirmation
Additional confirmation using PICs
PIC scan for peptide
marker
FFVAPFPEVFGK
found in Casein S1
Matrix: White wine

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Peptide Identification & Confirmation
Additional confirmation using PICs
PIC scan for peptide
marker
FFVAPFPEVFGK
found in Casein S1
Matrix: Red wine

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Compound name: YLGYLEQLLR (casein S1)
Correlation coefficient: r = 0.995512, r^2 = 0.991045
Calibration curve: 59440.7 * x + -3078.23
Response type: External Std, Area
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc
0 10 20 30 40 50 60 70 80 90 100
Response
0
1000000
2000000
3000000
4000000
5000001
Compound name: ALNEINQFYQK (casein S2)
Correlation coefficient: r = 0.995324, r^2 = 0.990670
Calibration curve: 4296.6 * x+ -289.732
Response type: External Std, Area
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc
0 10 20 30 40 50 60 70 80 90 100
Response
0
100000
200000
300000
Compound name: FALPQYLK (casein S2)
Correlation coefficient: r = 0.996969, r^2 = 0.993948
Calibration curve: 62929.5 * x + 567.125
Response type: External Std, Area
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc
0 10 20 30 40 50 60 70 80 90 100
Response
0
1000000
2000000
3000000
4000000
5000001
Compound name: GGLEPINFQTAADQAR (ovalbumin)
Correlation coefficient: r = 0.992720, r^2 = 0.985493
Calibration curve: 26267.2 * x + -5862.54
Response type: External Std, Area
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc
0 10 20 30 40 50 60 70 80 90 100
Response
0
500000
1000000
1500000
2000000
2500000
Matrix-match calibration curves

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Software Tools for Long-Term
Routine Allergens Analysis

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Conclusions
 Existing allergens methods employ ELISA & PCR-based techniques
 Recent years interest in tandem quad MS
– Increased selectivity
– Potential for multi-allergen analysis
– Capability to modify LC-MS methods for challenging matrices / proteins
affected during the food processing
 Routine methods need MRMs to be selective and sensitive to
identify and confirm the presence / absence allergenic protein(s).
 Method development stage can be time-consuming & useful to
have additional tools to support process
– RADAR – parallel acquisition of full scan and MRM data
– PICS – additional peptide confirmation
– TrendPlot – long term monitoring: QC standards, samples…

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Acknowledgements
CER Group
o Nathalie Gillard
o Olivier Spee
o Philippe Delahaut
Waters
Corporation
o Lee Gethings
o Kelly McMahon