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The Analysis of Allergens in Raw and Roasted Peanuts using Nanoscale UPLC & Time of-Flight Mass Spectrometry - Waters Corporation Food Research

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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 - Waters Corporation Food Research

  1. 1. ©2015 Waters Corporation 1 The Analysis Of Allergens in Raw and Roasted Peanuts using nanoscale UPLC & Time-of-Flight Mass Spectrometry
  2. 2. ©2015 Waters Corporation 2 Presentation Overview  Allergens Background  Technologies for Allergen Analysis  Experimental Workflow – Sample Prep – Instrument set-up – Software  Results  Conclusions
  3. 3. ©2015 Waters Corporation 3 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
  4. 4. ©2015 Waters Corporation 4 Background: Allergens Type of Food Categories
  5. 5. ©2015 Waters Corporation 5 Analysis of Allergens Technologies ELISA PCR MS IncreasingCurrentUsage IncreasingInstrument Complexity&Price
  6. 6. ©2015 Waters Corporation 6 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
  7. 7. ©2015 Waters Corporation 7 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)
  8. 8. ©2015 Waters Corporation 9 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
  9. 9. ©2015 Waters Corporation 10 nanoACQUITY data Raw peanut sample
  10. 10. ©2015 Waters Corporation 11 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
  11. 11. ©2015 Waters Corporation 12  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
  12. 12. ©2015 Waters Corporation 13 Advantages of MSE with PLGS Time-aligned spectra Low energy fragmentation High energy fragmentation *
  13. 13. ©2015 Waters Corporation 14 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
  14. 14. ©2015 Waters Corporation 15 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
  15. 15. ©2015 Waters Corporation 16 Peptide Sequence Identification: GSEEDITNPINLR
  16. 16. ©2015 Waters Corporation 17 Peptide sequences observed in both raw & roasted samples
  17. 17. ©2015 Waters Corporation 18 Relative intensities of peptide sequences present in both samples
  18. 18. ©2015 Waters Corporation 19 Peptide Coverage Raw and Roasted Samples Signal peptide: 1-25 Raw Sample Roasted Sample
  19. 19. ©2015 Waters Corporation 20 Quantification of Ara H1 in Matrix
  20. 20. ©2015 Waters Corporation 21 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
  21. 21. ©2015 Waters Corporation 22 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)
  22. 22. ©2015 Waters Corporation 23 Sample A – 1 : 3 dilution MS – Low collision energy data MSE – High collision energy data
  23. 23. ©2015 Waters Corporation 24 Sample B – 1 : 200 dilution MS – Low collision energy data MSE – High collision energy data
  24. 24. ©2015 Waters Corporation 25 Sample B – 1 : 200 dilution Peptide Coverage
  25. 25. ©2015 Waters Corporation 26 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

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