Detection of Cereal Detection of Cereal Proteins and DNA Using Proteins and DNA Using

MS, ELISA, and PCRMS, ELISA, and PCR

William J. Hurkman, Ph. D.William J. Hurkman, Ph. D.

USDA/ARSUSDA/ARS

Western Regional Research CenterWestern Regional Research Center

Albany, CAAlbany, CA

Wheat Flour ProteinsWheat Flour Proteins

Dough

Gluten

Glutenins Gliadins

Wash out starch granules

Mix with alcohol/water

SolubleInsoluble

Gliadins and Glutenins Are ProlaminsGliadins and Glutenins Are Prolamins

Barley, rye, and oat have proteins that are similar to wheat gliadins.

CerealCereal SpeciesSpecies NameName

WheatWheat TriticumTriticum Gliadins Gliadins BarleyBarley HordeumHordeum HordeinsHordeinsRyeRye SecaleSecale Secalins Secalins OatOat AvenaAvena AveninsAvenins

• Mass Spectrometry (MS)Mass Spectrometry (MS)• Monoclonal Antibodies (ELISA)Monoclonal Antibodies (ELISA)• Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)

Several analytical tools are currently Several analytical tools are currently available to detect the presence of cereal available to detect the presence of cereal prolamins in food products:prolamins in food products:

Detection MethodsDetection Methods

Endospermproteins

Spot Matching,Quantification,Profiling

2-DE

Gel Spots

Wheat EndospermProtein Database O-MALDI MS: Peptide mass mapping

Tandem MS: Peptide sequencing

DigestPro

Trypticfragments

MS Protein IdentificationMS Protein Identification

Sciex QSTAR

m/z1000 3000

Inte

nsity

20

0

95

2.5

0

84

2.5

1

16

46

.9

19

10

.910

45

.6

12

61

.6

10

61

.61

02

3.6

16

27

.9

86

6.4

5

22

11

.1

28

62

.3

17

57

.0

20

56

.0

15

15

.8

23

71

.1

13

20

.7

10

13

.6

10

90

.6

11

84

.6

15

67

.8

19

24

.9

20

97

.82

08

1.8

16

74

.9

26

66

.1

C:\Knexus\Data\000725_a\Unk_B101_10d_Spot_142_7\1SRef\pdata\1\1r. (12:33 08/31/00)

Data

WheatGrain

4.5 9.0 StdspI

Wheat Endosperm Wheat Endosperm ProteinsProteins

Albumins & Globulins(KCl-soluble)

pI

200.0116.3

66.355.4

36.531.021.5

14.4

MW(kD)

Gluten Proteins(KCl-Insoluble)

4.5 9.0 StdspI

HMW-GSHMW-GS GliadinsGliadins

αα, , γγ GliadinsGliadinsLMW-GSLMW-GS

97.4

αα-Amylase/trypsin-Amylase/trypsininhibitorsinhibitors

PeroxidasePeroxidaseSERPINSERPIN GAPDHGAPDH

• Many proteins can be separated Many proteins can be separated simultaneously.simultaneously.

• Protein identification is relatively rapid.Protein identification is relatively rapid.• Proteins can be quantified.Proteins can be quantified.• Proteins useful for antibody production.Proteins useful for antibody production.

• Limited to most abundant proteins.Limited to most abundant proteins.• Technically demanding.Technically demanding.• Equipment is expensive.Equipment is expensive.

ProsPros

ConsCons

MS Protein IdentificationMS Protein Identification

ELISAELISA

• Takes advantage of the ability of Takes advantage of the ability of antibodies to recognize and bind to antibodies to recognize and bind to proteins (antigens).proteins (antigens).

• More rapid and less expensive than More rapid and less expensive than mass spectrometry.mass spectrometry.

• Highly specific through use of Highly specific through use of monoclonal antibodies.monoclonal antibodies.

(Enzyme-Linked ImmunoSorbant Assay)

Monoclonal Antibody ProductionMonoclonal Antibody Production

Myeloma cells

Antigen is injected into a mouse to stimulate production of antibodies.

Fuse lymphocytes with myeloma cells to produce antibody producing hybrid cells.

Antibody

Lymphocytes from spleen

Screen for hybrid cell that produces specific antibodies and clone to produce unlimited quantities of monoclonal antibody.

Sandwich ELISASandwich ELISA

Sample & Stds Conjugate

Substrate

Inc. Inc.

Inc.

Wash

Wash

Stopping Reagent

+

-

Reactive Protein ConjugateR5 Antibody Substrate

or

• Detects:Detects:• Wheat gliadinsWheat gliadins• Barley hordeinsBarley hordeins• Rye secalinsRye secalins

• But not in oat avenins.But not in oat avenins.

R5 Monoclonal AntibodyR5 Monoclonal Antibody

Méndez et al. Eur. J. Gastroenterol. (2003) 15:465.Méndez et al. Eur. J. Gastroenterol. (2003) 15:465.

MKTFLIFVLLAMAMKIATAARELNPSNKELQSPQQSFSYQQQPFPQQPYPQQPYPSQQPYPSQQPFPTPQQQFPEQSQQPFTQPQQPTPIQPQQPFPQQPQQPQQPFPQPQQPFPWQPQQPFPQTQQSFPLQPQQPFPQQPQQPFPQPQLPFPQQSEQIIPQQLQQPFPLQPQQPFPQQPQQPFPQPQQPIPVQPQQSFPQQSQQSQQPFAQPQQLFPELQQPIPQQPQQPFPLQPQQPFPQQPQQPFPQQPQQSFPQQPQQPYPQQQPYGSSLTSIGGQ

R5 Monoclonal Antibody R5 Monoclonal Antibody Recognizes QQPFPRecognizes QQPFP

Repetitive sequence in gliadins, hordeins, and secalins, but not in avenins.

glutamine-glutamine-proline-phenylalanine-prolineglutamine-glutamine-proline-phenylalanine-proline

R5 ELISA ProsR5 ELISA Pros

• Detects wheat, barley, and rye prolamins. • Works well for a variety of unprocessed and

heat-processed foods. • Relatively rapid (1 h 30 min).• Sensitive (1.5 ppm).• Available in commercial kits.

• Tested in 20 laboratories (results soon to be published).

• Temporarily endorsed by the Committee on Methods of Analysis and Sampling of the Codex Alimentarius Commission (more data needed).

R5 ELISA ConsR5 ELISA Cons

•Does not distinguish between wheat, barley, and rye.

•Detects only gliadins in wheat.

•Celiac patients also react to high and low molecular weight glutenins.

•Some ingredients have glutenins, but not gliadins.

PCR (Polymerase Chain Reaction) AmplificationPCR (Polymerase Chain Reaction) Amplification

Denature TemplateDNA

Anneal Primers(Forward & ReverseOligonucleotides)

Extend DNA(DNA polymerase,

DNTPs)

PCR Primers for ProlaminsPCR Primers for Prolamins

Wheat Gliadin (AF280605m, 2315-2495)....cagaaa gcgagtggaa agatgaaagc aagccatgcg atgaaaacta taaagacaca tgcaccacca aggccacctt ccatcatcca aacttcacac acctagacca caagcatcaa aggcaagcaa gcagtagtca ccacaaatcc aacatgaaga ccttcctcat ctttgtcctc cttgc.....

Rye Secalin (X60295, 325-505)....tttttc agaaagcgag ttcaatgatg aatgaatcca taccataaca actataaata cacatgcacc attatagtca ccttccatca tccaaacttc acgcaccaag atcagaaaca tcaattccaa gcaagcatta gtaaccacaa atccaacatg aagaccttcc tcatctttgt cctcg.....

Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349.

PCR Primers for ProlaminsPCR Primers for Prolamins

Barley Hordein (X03103, 1618-1763).......att aattcccaaa ctgaacgact acgtgaaaag acagtcacac catgtttgta catccacccc tttgctcgaa atggcgttct tttgctggac agccgagctt cagaatctgc cgtcaagttc ctgagatcca tccacagatg tcgttcacat tgttcgccat g.........

Oat Avenin (J05486 1097-1200)......cgct cagtggcttc taagaacact acaagagcta tagtactaca taaataccat cagcgtttag ccgatggacc gatcttgtag cggtgacaaa taaaataaaa ..........

Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349.

PCR SpecificityPCR Specificity

Winter WheatWinter Wheat ++ -- -- --Spring WheatSpring Wheat ++ -- -- --Durum WheatDurum Wheat ++ -- -- --SpeltSpelt ++ -- -- --

KamutKamut ++ -- -- --

TriticaleTriticale ++ -- -- --RyeRye -- ++ -- --BarleyBarley -- -- ++ --OatOat -- -- -- ++

Primer PairsSpecies Wheat Rye Barley Oat

Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349.

PCRPCR

• Species specific: distinguishes between Species specific: distinguishes between wheat, rye, barley, and oats.wheat, rye, barley, and oats.

• Sensitive and rapid.Sensitive and rapid.• Compliments ELISA results.Compliments ELISA results.

• Detects DNA rather than actual protein. Detects DNA rather than actual protein.

ProsPros

ConsCons

SummarySummary

• Mass spectrometryMass spectrometry • Excellent tool for protein identification.Excellent tool for protein identification.• Sensitive, but not rapid.Sensitive, but not rapid.• Expensive and technically demanding.Expensive and technically demanding.

• ELISAELISA• Works well for a variety of unprocessed and heat-

processed foods. • Sensitive and rapid.

• PCRPCR• Species specific.Species specific.• Compliments ELISA.Compliments ELISA.• Sensitive and rapid.Sensitive and rapid.