Development of Complementary Mass Spectrometry Methods for Screening Allergens in Food in ifaam (matrix: chocolate dessert)

Transcription

1 Universität für Bodenkultur Wien Interuniversitäres Department für Agrarbiotechnologie Tulln Development of Complementary Mass Spectrometry Methods for Screening Allergens in Food in ifaam (matrix: chocolate dessert) SYMPOSIUM Frontiers in Food Allergy and Allergen Risk Assessment and Management 18th 20th April 2018 Madrid, Spain Kathrin Lauter, M.Sc. University of Natural Resources and Life Sciences Vienna, Dept. IFA-Tulln, Centre for Analytical Chemistry Konrad Lorenz Straße 20 A-3430 Tulln, Austria 1 Bezeichnung deutsch: ev. Untereinheit 2. Ebene I ev. Untereinheit 3. Ebene I Univ. Prof. DI. Dr. Max Mustermann

2 Complementary multi-allergen Method - LC-MS/MS Food matrices Chocolate bar (PEI) Chocolate dessert (BOKU) Cookies (JRC) Allergenic ingredients Skimmed milk powder Skimmed egg white powder Peanut flour Hazelnut flour Walnut flour 2

3 Protein MS methods Graph adjusted from: J. Proteome Res., 2013, 12 (3), pp , DOI: /pr301201x 3

4 MS/MS sample extraction proteins digest peptides chromatographic separation liquid chromatography 4

5 Set up a screening method Allergen Jug r Jug r Jug r 3 Jug r Cor a Cor a Cor a Cor a Cor a Cor a Cor a Ara h 1 Ara h 2 Ara h 3/4 Ara h 5 Ara h Gal d 1 Gal d 2 Gal d 4 Gal d 3 Uniprot- Accession P93198 Q9SEW4 C5H617 Q2TPW5 Q9ATH2 Q8W1C2 Q9FSY7 Q8S4P9 Q84T21 Q84T91 D0PWG2 P43238 Q6PSU2 Q8LKN1 D3K177 Q647G9 P02754 P00711 P02662 P02663 P02666 P02668 P01005 P01012 P00698 P02789 Targeted MRM-method (for screening) 5 25 AAs No posttransl. modifications No missed cleavages No Methionine All y-ions 210 peptides Dwelltime: 5 ms 5

6 MS-instrument Solvent A: H2O with 0.1% (v/v) FA Sovent B: MeOH with 0.1% (v/v) FA Flow: 0.3 ml/min gradient for separation (10-75% B) LC system: Agilent 1290 Infinity Binary LC System (Agilent) LC guard column: SecurityGuard ULTRA Cartrige UHPLC C18- Peptide for 2.1 mm ID Columns (Phenomenex) LC column: Aeris PEPTIDE 3.6u XB C18 column 150*2.1 mm (Phenomenex) MS: QTRAP 6500 System (Sciex) equipped with an IonDrive TM Turbo V source Software: Skyline (MacCoss Lab Software) 6

7 Extraction Optimisation Stage 1: Buffer 50 mm Tris-HCl buffer, ph mm ammonium bicarbonate buffer, ph 7.8(ABC 50 ) 100 mm ammonium bicarbonate buffer, ph 8.1 (ABC 100 ) Tris-Borat buffer, ph 8.5 (8 mm Tris, 10 mm (NH 4 ) 2 B 10 O 16 ) Stage 2: Detergents No detergent 5 % (w/v) Sodium deoxycholate (SDC) 0.6 M Urea 6 M Urea Stage 4: Time/Temperature Combinations RT for 4 h (standard) 60 C for 15 min (waterbath) 60 C for 30 min (waterbath) Stage 5: sample-buffer ratio 1:5, 1:10, 1:20 7

8 What is best? Decision based on: protein content measurement (2D-Quant Kit) Found peptides per allergenic ingredient Peak intensities ABC 50 ABC100 Tris-HCl TBB PN WN HN SMP EWP Total Number of peptides found 8

9 Peak intensities multiallergen method = best compromise 9

10 Tryptic Digest Optimisation Stage A: Trypsin brand Trypsin, Proteomics Grade, BioReagent, Dimethylated (Sigma # T6567) Trypsin, Sequencing Grade Modified (Promega # V5111) Stage B: protease:protein ratio 1:5 1:10 1:25 1:50 1:100 Stage C: Temperature 37 C 55 C Stage D: Detergents No detergent 0.1% RapiGest SF 0.04% RapiGest SF 0.5% Sodium deoxycholate (SDC) 10% Acetonitrile (ACN) 10

11 What is best? Proteins and number of peptides per allergenic ingredient Peak intensities Tryptic digest Optimisation - Stage C 120 SMP 12 SMP 11 SMP 10 number of peptides SMP 9 SMP 6 SMP 5 SMP Bod d 4 HN Cor a 12 HN Cor a 11 HN Cor a 10 HN Cor a 9 EWP Gal d 4 EWP Gal d 3 EWP Gal d 2 WN Jug r C_1:5 37 C_1:10 55 C_1:5 55 C_1:10 WN Jug r 2 PN Ara h 5 PN Ara h 3/4 PN Ara h 1 11

12 Tryptic Digest Optimisation Walnut Peanut 4,00E+06 8,00E+06 3,00E C_1:5 37 C_1:10 6,00E C_1:5 37 C_1:10 peak area (cps) 2,00E C_1:5 55 C_1:10 peak area (cps) 4,00E C_1:5 55 C_1:10 1,00E+06 2,00E+06 0,00E+00 peptides 0,00E+00 peptides Hazelnut peak area (cps) 1,20E+07 1,00E+07 8,00E+06 6,00E+06 4,00E+06 2,00E C_1:5 55 C_1:5 37 C_1:10 55 C_1:10 0,00E+00 peptides 12

13 Tryptic Digest Optimisation Skimmed egg white powder 8,00E+06 6,00E C_1:5 55 C_1:5 37 C_1:10 55 C_1:10 peak area (cps) 4,00E+06 2,00E+06 0,00E+00 peptides Skimmed milk powder 2,00E+07 1,50E C_1:5 55 C_1:5 37 C_1:10 55 C_1:10 peak area (cps) 1,00E+07 5,00E+06 0,00E+00 peptides 13

14 Final Choice of Sample Protocoll Extraction Tryptic Digestion Tris borate buffer + 6 M urea 5 µg of protein in 60 µl ABC 50 1:10 DTT for 15 min at 60 C 60 C for15 min IAA for 15 min at RT (dark) 30 min at 16,100xg at 4 C Ad trypsin (1:5 proteaseto-protein ratio) 16 h at 37 C incubation Stop with formic acid 14

15 Chioce of peptides for MS-Parameter optimisation Skimmed milk powder 1,00E+07 8,00E+06 R R R 6,00E+06 4,00E+06 2,00E+06 R X R R R 0,00E+00 FFVAPFPEVFGK VLPVPQK YLGYLEQLLR ALCSEK AVPYPQR NAVPITPTLNR YIPIQYVLSR FALPQYLK VIPYVR IDALNENK EDVPSER GLDIQK VNELSK ALNEINQFYQK IPAVFK CEVFR LVVSTQTALA ITVDDK DDSPDLPK LDQWLCEK SPAQILQWQVLSNTVPAK TPEVDDEALEK LTEEEK LVTDLTK AEFVEVTK Bod d Bod d Bod d ,E+07 8,E+06 peak area 6,E+06 4,E+06 2,E+06 0,E+00 4/02 4/03 4/01 4/04 5/04 5/02 5/01 5/03 6/01 9/03 9/02 9/01 9/04 10/03 10/01 10/02 11/02 11/01 11/03 12/02 12/01 0 ppm 3 ppm 10 ppm 30 ppm 50 ppm 100 ppm 15

16 Final peptide list 26 peptides Allergen Ara h 1 Ara h 2 Ara h 3 Ara h 3/4 Ara h 3/4 Jug r 2 Jug r 2 Jug r 4 Cor a 9 Cor a 9 Cor a 9 Cor a 11 Gal d 2 Gal d 2 Gal d 2 Gal d 2 Gal d 3 Gal d Peptide Sequence DLA NLP TAN WLG FFV ATL EGV ALP TIE INT ALP LLS DIL LYA GGL HIA AQS YFG IPA IDA FFV YLG NAV FAL VLP AVP 16

17 Material: chocolate dessert ppm [protein of allergenic ingredient/chocolate dessert] Blank = considered as a sample, necessairy to determine statistical limits of measurements like LOD and LOQ 5 samples which covers the minimum number of materials 17

18 Set-up: Extraction Tryptic Digest MS-analysis TRIPLICATE Digest 1 M1 Extraction 1/2/3 (0, 3, 10, 30, 100 ppm) Digest 2 M1 Digest 3 M1 M2 M2 M2 M3 M3 M3 A single extract of each level on three seperate days 18

19 Relative quantification with isotopically labelled peptides Heavy labelled peptides (SpikeTides_TQL) 13 C and 15 N labelled chemical Qtag (cleavable by trypsin) spiked into samples at constant level Heavy:light peptide ratio 19

20 Peptide LOD ppm LOQ ppm Ara h Ara h Ara h 3/ Ara h 3/ Ara h 3/ Jug r Jug r Jug r Cor a Cor a Cor a Cor a Gal d Gal d Gal d Gal d Gal d Gal d Reference dose (mg of protein) VITAL 2.0 Action level 1 50 g serving size 10 g serving size 5 g serving size 0.2 < 4 ppm < 20 ppm < 40 ppm no data no data no data no data 0.1 < 2 ppm < 10 ppm < 20 ppm 0.03 < 0.6 ppm < 3 ppm < 6 ppm 0.1 < 2 ppm < 10 ppm < 20 ppm LLLLLL = AAAAAAAAAAAAAA oooo bbbbbbbbbb mmmmmmmmmmmm + 3 SSSSSSSSSSSSSSSS DDDDDDDDDDDDDDDDDD LLLLLL = AAAAAAAAAAAAAA oooo bbbbbbbbbb mmmmmmmmmmmm + 10 SSSSSSSSSSSSSSSS DDDDDDDDDDDDDDDDDD ppm mg protein of allergenic ingredient / kg food matrix 20

21 Peptide LOD ppm LOQ ppm Ara h Ara h Ara h 3/ Ara h 3/ Ara h 3/ Jug r Jug r Jug r Cor a Cor a Cor a Cor a Gal d Gal d Gal d Gal d Gal d Gal d Reference dose (mg of protein) VITAL 2.0 Action level 1 50 g serving size 10 g serving size 5 g serving size 0.2 < 4 ppm < 20 ppm < 40 ppm no data no data no data no data 0.1 < 2 ppm < 10 ppm < 20 ppm 0.03 < 0.6 ppm < 3 ppm < 6 ppm 0.1 < 2 ppm < 10 ppm < 20 ppm Multi-allergen method Still just complementary in terms fitness for VITAL 2.0 Compromise in sample preparation for egg and milk better LOD/LOQ might be possible with a different extraction protocol Is a unique extraction protocol the future? ppm mg protein of allergenic ingredient / kg food matrix 21

22 Clare Mills (UniMan) Ivona Baricevic (Uniman) Victoria Lee (Uniman) Rebekah Sayers (Uniman) Chiara Nitride (Uniman) Andreas Reuter (PEI) Thomas Schulenborg (PEI) Gavin O Connor (JRC) Sabine Baumgartner (BOKU) Frontiers in Food Allergy and Allergen Risk Assessment and Management, 18th-20th April 22