analysis of milk and egg allergens in wine using uplc-ms · 2012-10-03 · analysis of milk and egg...
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©2012 Waters Corporation 2
Analysis of Milk and Egg Allergens in Wine Using UPLC-MS
Antonietta Gledhill Senior Market Development Manager
Work performed in collaboration with:
©2012 Waters Corporation 3
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
©2012 Waters Corporation 4
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
©2012 Waters Corporation 8
Analysis of Allergens Popular Technologies Adopted
ELISA
PCR
MS Incr
easi
ng
Cu
rren
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sag
e
Incr
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ng
In
stru
men
t C
om
ple
xity
& P
rice
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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…
NN
PFYF
PSR
DLA
FPG
SG
EQVEK
VLL
EEN
AG
GEQ
EER
ISM
PVN
TPG
QFE
DFF
PASSR
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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
©2012 Waters Corporation 19
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
©2012 Waters Corporation 21
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
Cas
ein
S1-
YLG
Cas
ein
S1-
FFV
Ova
lbum
in-
EVV O
valb
umin
-G
GL Ova
lbum
in-
DIL
O
valb
umin
-EL
I
Cas
ein
S2-
FAL
Cas
ein
S1
- VPQ
Cas
ein
S2-
ALN
4
3
2 1
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
©2012 Waters Corporation 34
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 = 0.991045Calibration curve: 59440.7 * x + -3078.23Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc0 10 20 30 40 50 60 70 80 90 100
Res
pons
e
0
1000000
2000000
3000000
4000000
5000001
Compound name: ALNEINQFYQK (casein S2)Correlation coefficient: r = 0.995324, r^2 = 0.990670Calibration curve: 4296.6 * x + -289.732Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc0 10 20 30 40 50 60 70 80 90 100
Res
pons
e
0
100000
200000
300000
Compound name: FALPQYLK (casein S2)Correlation coefficient: r = 0.996969, r^2 = 0.993948Calibration curve: 62929.5 * x + 567.125Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc0 10 20 30 40 50 60 70 80 90 100
Res
pons
e
0
1000000
2000000
3000000
4000000
5000001
Compound name: GGLEPINFQTAADQAR (ovalbumin)Correlation coefficient: r = 0.992720, r^2 = 0.985493Calibration curve: 26267.2 * x + -5862.54Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Conc0 10 20 30 40 50 60 70 80 90 100
Res
pons
e
0
500000
1000000
1500000
2000000
2500000
Matrix-match calibration curves
©2012 Waters Corporation 36
Software Tools for Long-Term Routine Allergens Analysis
©2012 Waters Corporation 37
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…
©2012 Waters Corporation 38
Acknowledgements
CER Group o Nathalie Gillard
o Olivier Spee
o Philippe Delahaut Waters Corporation o Lee Gethings
o Kelly McMahon