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

©2015 Waters Corporation 1

Discovery and Analysis of Peanut Allergens

using Proteomic Approaches with Ion

Mobility and High Resolution Mass

Spectrometry


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


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)


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

http://guangdonginspection.com/blog/wp-content/uploads/2013/09/Preventive-corrective-action.png


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


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 )


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


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


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


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)


Extraction Methodology Comparison

Provided by Phil Johnson, Anuradha Balasundaram, Rebekah Sayers, Justin Marsh and Clare Mills (University of Manchester)


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


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


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]


Increasing system peak capacity by ion mobility separation (IMS)

CID

TRAP

ION MOBILITY SEPARATION

TRANSFER

HELIUM CELL

Drift time

m/z


Ion mobility: introduction

Ion mobility leads to separation based on molecular conformation

030709_CYTOTEST2_G2.raw : 1

Time0.00 2.00 4.00 6.00 8.00 10.00 12.00

%

0

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


Ionized

Precursors

Precursors

Transferred to TOF

MS

UPLC/HDMSE …deconvoluting chimericy

Co-Eluting

Peptides


Ionized

Precursors

Precursors &

Products Time

Aligned

UPLC/HDMSE …deconvoluting chimericy

Co-Eluting

Peptides


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


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)


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


‘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


Label free protein quant via the Waters method

Relative quantitation via comparison of normalised peak volumes - only been possible following introduction of reproducible nanoUPLC


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


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/µL 50

spike][Protein intensity peptide

interest] of[Protein intensity peptide

3

1i

3

1i

Conc =


Data processing – Progenesis QI-P

alignment

peak detection

identification

protein quantitation

statistics

peptide quantitation


Progenesis QI-P Workflow

27,313 features

Alignment

Normalization

Peak Picking

Database Searching Protein Output

(including label-free quantitation)


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%


Qualitative Overview – GPC flour (peanut allergen families)

Average # peptides

Summed (log) intensity of top3 peptides

Average % sequence coverage


Normalized Relative Abundance (dynamic range of the peanut proteome)

Nearly 4 orders of magnitude dynamic range


Relative Quantification (significant isoforms)

Ara h 1 Ara h 3 Ara h 10,11


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


From discovery to targeted…..


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


Acknowledgement

University of Manchester

– Phil Johnson

– Anuradha Balasundaram

– Rebekah Sayers

– Justin Marsh

– Clare Mills

Science

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