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Discovery and Analysis of Peanut Allergens using Proteomic approaches with Ion Mobility and High Resolution Mass Spectrometry - Waters Corporation Food Research

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Accurate data are needed on threshold doses for allergenic foods. Data are also needed to demonstrate that available methods of analysis can detect and quantify allergens in foods at or around these threshold levels and that they are robust and fit for purpose. This is of major concern as no curative treatment is currently available for food allergy and accidental ingestion of the culprit food can lead to severe clinical symptoms. Elimination of the problem food ingredient from the diet reduces the risk of allergic reactions but this can lead to other issues such as deficiencies, eating disorders and growth retardation. Emergency medication is available including Antihistamines (H1 blockers), EpiPen (adrenaline-autoinjector) and Corticosteroids. This presentation describes investigations into peanut allergens and their quantitation in highly complex samples.

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Discovery and Analysis of Peanut Allergens using Proteomic approaches with Ion Mobility and High Resolution Mass Spectrometry - Waters Corporation Food Research

  1. 1. ©2015 Waters Corporation 1 Discovery and Analysis of Peanut Allergens using Proteomic Approaches with Ion Mobility and High Resolution Mass Spectrometry
  2. 2. ©2015 Waters Corporation 2 Contents  General Introduction – Food allergy – Regulatory aspects  Extraction Methods – RapiGest protocol  LC/MS Analysis – DIA strategy (HDMSE) – Label-free quantification – Progenesis QI-P  Conclusions Drift time m/z
  3. 3. ©2015 Waters Corporation 3 Immunological Aspects of Food Allergy  Food allergic reaction is an IgE mediated reaction to specific food proteins – Prevalent in c. 2% of the adult and 8% of child population – Symptoms can range from mild to severe (life-threatening)
  4. 4. ©2015 Waters Corporation 4 Food Allergy – avoidance & preventative actions?  No curative treatment is available for food allergy  Accidental ingestion of the culprit food can lead to severe clinical symptoms – Elimination diet o Reduce the risk of allergic reactions o Disadvantages: deficiencies, eating disorders, growth retardation – Emergency medication o Antihistamines (H1 blockers) o EpiPen (adrenaline-autoinjector) o Corticosteroids Preventative actions? Effective tools for detection & quantitation are needed for effective labelling
  5. 5. ©2015 Waters Corporation 5 EU perspective – Statutory Food Labelling Laws  The rules for pre-packed foods establish a list of 14 food allergens, which must be indicated by reference to the source allergen whenever they, or ingredients made from them, are used at any level in pre-packed foods, including alcoholic drinks  Labelling rules in European Directives 2003/89/EC & 2006/142/EC ensure that all consumers are given comprehensive ingredient listing information and make it easier for people with food allergies to identify ingredients they need to avoid  Food Information for Consumers Regulation (EU) No. 1169/2011 builds on current allergen labelling provisions for pre-packed foods & introduces a new requirement for allergen information to be provided for foods sold non-packed or pre-packed for direct sale – Allergen labelling rules will be changing in December 2014
  6. 6. ©2015 Waters Corporation 6 Allergen Classification EU 14 major priorities Cereals containing gluten, crustaceans, molluscs, eggs, fish, peanuts, nuts, soybeans, milk, celery, mustard, sesame, lupin and sulfur dioxide (at levels >10mg/kg or 10 mg/litre, expressed as SO2 )
  7. 7. ©2015 Waters Corporation 7 Establishment of Threshold Doses  Threshold dose establishment – ongoing research activity – Safety assessment LOAEL or NOAEL  Commission Regulation (EC) No. 41/2009 established levels of gluten for foods claiming to be either 'gluten-free' or 'very low gluten‘ (January 2012) – 'gluten-free': at 20 parts per million of gluten or less – 'very low gluten': at 100 parts per million of gluten or less - however, only foods with cereal ingredients that have been specially processed to remove the gluten may make a 'very low gluten' claim  These regulations apply to all foods, pre-packed or sold loose, such as in health food stores or in catering establishments
  8. 8. ©2015 Waters Corporation 8 Sample preparation strategy  Solubilise and extract protein using aq buffer from complex matrix  Protein denaturation using detergents & chaotrophic agents (RapiGest™) to linearise the 3D structure  Proteolytic digestion using trypsin to cleave the protein into reproducible and peptide sequences (6 – 12 amino acids)  Additional sample clean-up & enrichment – SPE – Immuno-affinity column using specific anti-peptide IgG  Filtration & dilution in mobile phase A prior to LC-MS analysis
  9. 9. ©2015 Waters Corporation 9 Example Extraction Protocol Sonicate in 60⁰C heated ultrasonic water bath for 15 min/vortex every 5 min Extraction Buffer 50mM Tris.HCl pH 8.8, 50mM DTT & 0.04% Rapigest High speed centrifugation (10000rpm,10min, RT) Collect the supernatant and store at -20⁰C prior to analysis RapiGest™ SF is a reagent used to enhance enzymatic digestion of proteins RapiGest SF helps solubilize proteins, making them more susceptible to enzymatic cleavage without inhibiting enzyme activity
  10. 10. ©2015 Waters Corporation 10 SDS-PAGE (peanut flour extracts) Lane Sample A Raw B Raw C Raw D Roasted E Roasted F Roasted Provided by Phil Johnson, Anuradha Balasundaram, Rebekah Sayers, Justin Marsh and Clare Mills (University of Manchester)
  11. 11. ©2015 Waters Corporation 11 Extraction Methodology Comparison Provided by Phil Johnson, Anuradha Balasundaram, Rebekah Sayers, Justin Marsh and Clare Mills (University of Manchester)
  12. 12. ©2015 Waters Corporation 12 How do we deal with this complexity?  Increase ‘peak capacity’ of analytical system  Separate analytes before ID with MS/MS – LC dimension o UPLC o Multi-dimension LC – MS dimension o Mass resolution o Ion mobility  Use strategies which will work with multiplex spectra – LC-MSE / LC-HDMSE (data-independent acquisition) – Bateman et al. JASMS (2002);13:792 – Advocacy increasing “some form of multiplexing of MS/MS in high resolution format will most likely need to be a component of future shotgun proteomics strategies” Michalski, Cox, Mann. JPR (2011);10:1785
  13. 13. ©2015 Waters Corporation 13 Why DIA and not DDA?  Only most intense peptides are fragmented  Only most intense peptides can be identified  These may not be the peptides of interest  Other peptides may be eluting while in MS/MS mode  Not fragmented, not identified  These may be the peptides of interest  Quantification is difficult  Cannot use survey data as we do not sample peak effectively  No survey data is being collected while in MS/MS mode  Run-to-run reproducibility is poor
  14. 14. ©2015 Waters Corporation 14 2 IMS Increases Peak Capacity: The Datacube  Peak capacity = NLC x NIM x Nm/z  BUT: LC, m/z and IM not completely orthogonal  BUT: datacube non-uniformly populated  Nm/z > NLC > NIM [10,000’s > 1000’s (2D-LC) or 100’s (1D-LC) > 10’s]
  15. 15. ©2015 Waters Corporation 15 Increasing system peak capacity by ion mobility separation (IMS) CID TRAP ION MOBILITY SEPARATION TRANSFER HELIUM CELL Drift time m/z
  16. 16. ©2015 Waters Corporation 16 Ion mobility: introduction  Ion mobility leads to separation based on molecular conformation 030709_CYTOTEST2_G2.raw: 1 100 0.00 2.00 4.00 6.00 8.00 10.00 12.00 % 0 100 Cytochrome C +8 charge Synapt G2 Separation of isobaric ions Isobaric ions Drift time m/z
  17. 17. ©2015 Waters Corporation 17 Ionized Precursors Precursors Transferred to TOF MS UPLC/HDMSE …deconvoluting chimericy Co-Eluting Peptides
  18. 18. ©2015 Waters Corporation 18 Ionized Precursors Precursors & Products Time Aligned UPLC/HDMSE …deconvoluting chimericy Co-Eluting Peptides
  19. 19. ©2015 Waters Corporation 19 Concept of high-definition (HD)-MSE 19 Retention time aligned precursor and product ions Drift time aligned precursor and product ions ION MOBILITY SEPARATION DB SEARCH
  20. 20. ©2015 Waters Corporation 20 Experimental Details (LC/MS)  LC – nanoAcquity UPLC – Solvent A: Water/0.1% FA – Solvent B: ACN/0.1% FA – 300 nL/min flow rate – Trapping configuration (3 mins @ 5 µL/min) – 1 µL partial loop injection  MS – Synapt G2-Si – HDMSE acquisition mode (0.5s scan rate) – Resolution mode (25,000 resolution) – LE: 4 eV; HE: 19-45 eV – Lockmass correction: Glu-fibrinopeptide (m/z 785.8426)
  21. 21. ©2015 Waters Corporation 21 Main principles of quantitative ‘discovery proteomics’ using MS Mueller LN et al. JPR (2008);7:51 • ‘Labelled’ methods • Compare peak areas across peptide peak pairs separated by ‘tag’ mass • ‘Label-free’ methods • Label-free quant • Compare peptide peak volumes across LC-MS runs • Spectral counting • Compare number of MS/MS measurements for a peptide peak across LC-MS runs
  22. 22. ©2015 Waters Corporation 22 ‘Labelled’ vs ‘Label-free’  Label-free needs no sample modification / manipulation  Can be applied to any samples, including non-growing  No constraints on experimental designs  New samples can be compared to historical data  No reagent costs (iTRAQ is $400/sample!)1  No time for sample preparation reactions  No variability introduced due to preparation reactions 1.Dekkers DHW et al. Curr Proteomics (2010);7:108
  23. 23. ©2015 Waters Corporation 23 Label free protein quant via the Waters method Relative quantitation via comparison of normalised peak volumes - only been possible following introduction of reproducible nanoUPLC
  24. 24. ©2015 Waters Corporation 24 The Waters method also gives absolute quantification [ADH] = x mol [BSA] = x mol [HBA] = 0.5 x mol [HBB] = 0.5 x mol • Serendipitous discovery • Protein standard development work Silva JC et al. MCP (2006);5:144
  25. 25. ©2015 Waters Corporation 25 The Waters method also gives absolute quantification  The intensity response under electrospray conditions of the three most intense peptides is a function of the molar amount infused in the mass spectrometer – The estimated absolute amount of a protein can be calculated from the intensity of the top three ionizing tryptic peptides o Assumption is that the “best ionizing peptides” have similar chemical composition – The absolute amount can be calculated for every identified protein Silva JC et al. MCP (2006);5:144  fmol/µL50 spike][Proteinintensitypeptide interest]of[Proteinintensitypeptide 3 1i 3 1i      Conc =
  26. 26. ©2015 Waters Corporation 26 Data processing – Progenesis QI-P alignment peak detection identification protein quantitation statistics peptide quantitation
  27. 27. ©2015 Waters Corporation 27 Progenesis QI-P Workflow 27,313 features Alignment Normalization Peak Picking Database Searching Protein Output (including label-free quantitation)
  28. 28. ©2015 Waters Corporation 28 Processing/Search Parameters  Progenesis QI-P – Apex 3D processing parameters o Low energy threshold = 150 counts o High energy threshold = 30 counts o Lockmass correction = 785.8426 m/z (GFP) – IA database search parameters o Minimum fragment ions per peptide = 1 o Minimum fragment ions per protein = 3 o Minimum peptide per protein = 1 o Uniprot database (A. Hypogaea) reviewed sequences o False discovery rate = 4%
  29. 29. ©2015 Waters Corporation 29 Qualitative Overview – GPC flour (peanut allergen families) Average # peptides Summed (log) intensity of top3 peptides Average % sequence coverage
  30. 30. ©2015 Waters Corporation 30 Normalized Relative Abundance (dynamic range of the peanut proteome) Nearly 4 orders of magnitude dynamic range
  31. 31. ©2015 Waters Corporation 31 Relative Quantification (significant isoforms) Ara h 1 Ara h 3 Ara h 10,11
  32. 32. ©2015 Waters Corporation 32 Batch-to-batch consistency (GPC flour) Sequences showing greatest variation : all Ara h 3 with the exception of P43237 (Ara h 1) # flour batches analyzed = 7 (in triplicate) Batches 2 & 4
  33. 33. ©2015 Waters Corporation 33 From discovery to targeted…..
  34. 34. ©2015 Waters Corporation 34 Conclusions  Optimized protein extraction protocol – Using acid labile detergent (RapiGest) – Applicable to other categories of allergens  Comprehensive characterization of the peanut proteome  Achieved using a DIA approach utilizing ion mobility – Over 300 proteins identified with high % sequence coverage (per injection) – Label-free quantification of all identified proteins  Potential target peptides identified for absolute quantification
  35. 35. ©2015 Waters Corporation 35 Acknowledgement  University of Manchester – Phil Johnson – Anuradha Balasundaram – Rebekah Sayers – Justin Marsh – Clare Mills

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