Susan B. Altenbach, Frances M. Dupont, William H. Vensel, Charlene K. Tanaka,

Paul Allen & William J. Hurkman USDA-ARS Western Regional Research Center, Albany, CA USA

XIth International Gluten Workshop

August 12-15, 2012

Beijing, China

a critical step in understanding the effects of environment

on flour quality and immunogenic potential

Environment

Creating a detailed map of the

wheat flour proteome:

Genes

Quality

Fertilizer Temperature Drought

Proteome Transgenic Plants

• Challenges in creating a detailed map of the

wheat flour proteome

• Use of the map to identify changes in the flour

proteome that result from growth conditions

• Capturing discoveries from proteomics studies

to define roles of specific proteins in flour quality

and in the response to the environment

Outline

Total Flour Protein

SDS + DTT

Non-gluten Proteins

KCl-soluble, MeOH-insoluble

~11% of total

Non-gluten Proteins

KCl-soluble, MeOH-soluble

~5% of total

Gluten Proteins

KCl-insoluble

~80% of total

Gluten proteins

comprise ~80% of

flour protein

Separation of flour proteins by 2-DE

Peptides separated and further fragmented,

spectra generated

Identification of proteins by tandem mass

spectrometry (MS/MS)

Proteins digested with trypsin

Spectra matched to data generated

in silico from protein databases

MS/MS yields sequence information rather than just the mass of the peptides

• Proteins are digested by the protease into peptides

that are of a size suitable for MS/MS analysis.

• Representative protein sequences are found in the

database used to analyze spectra.

MS/MS Identification Requires That

Proteomic maps of non-gluten proteins

Wong et al.,Plant Cell Physiol. 45: 407-415, 2004.

metabolic proteins

structural proteins

defense proteins

a-amylase/trypsin inhibitors

defense proteins

KCl-soluble/MeOH-insoluble

KCl-soluble/MeOH-soluble

Vensel et al. Proteomics 5: 1594-1611, 2005.

Wheat gluten proteins present challenges

for MS/MS identification

• Identifications were based on very few peptides -

sequence coverage <10%

• Protein families were sometimes identified, but individual

proteins within each family could not be distinguished

• Many proteins were not identified at all

The major groups of gluten proteins contain many

similar sequences and there is considerable sequence

heterogeneity among different cultivars

Why??

Proteins are not readily digested with trypsin

Gluten protein sequence diversity is not

adequately reflected in current databases

Gluten proteins have very repetitive sequences that

are rich in glutamine and proline

% Gln + Pro

HMW-GS & LMW-GS 43-54%

alpha & gamma gliadins 49-56%

omega gliadins 68-73%

It is Important to Distinguish Individual Gluten

Proteins for Studies of Wheat Flour Quality

• Minor differences in protein sequence can result in

different functional properties (extra cysteine).

• Small differences in protein sequence can affect

potential to trigger celiac disease and food allergies.

Approach

• Digest each protein with three separate proteases,

generate spectra and combine data

• Optimize database by including sequences of gluten

proteins from the cultivar under study

Trypsin

Chymotrypsin

Thermolysin

Alpha Gliadins

136 ESTs assembled into 19 contigs

Analysis of ESTs from Butte 86

• 13 encoded full-length proteins

• One contained seven cysteines instead of six

• Eight contained known celiac epitopes

• Only two were perfect matches with alpha

gliadins in NCBI*

*167 alpha gliadins in NCBI

Altenbach et al., J. Cereal Sci., 52: 143-151. 2010.

Gamma Gliadins

153 ESTs assembled into 11 contigs

• 9 encoded full-length proteins

• Four contained nine cysteines instead of usual eight

• Only one was a perfect match with a gamma gliadin in

NCBI

*323 gamma gliadin sequences in NCBI

Altenbach et al., BMC Plant Biology 10:7. 2010.

“SuperWheat” Database

2,562,722 protein sequences

• NCBI non-redundant green plant protein sequences

• Proteins translated from:

- Contigs from wheat EST assemblies

(TaGI Release 10.0,TaGI Release 11.0, US Wheat

Genome Project, HarvEST 1.14, Unigene Build #55

- Butte 86 ESTs

- Butte 86 contigs

• Proteins in individual 2-DE spots cleaved with trypsin,

chymotrypsin, or thermolysin.

Identification of Gluten Proteins

from Butte 86 by MS/MS

• Spectra generated with QSTAR Pulsar i quadropole time-

of-flight mass spectrometer with nano-electrospray

source and nano-flow LC.

• Two search engines (Mascot and X!Tandem) were used

to interrogate the “Superwheat” database with spectra.

• Results were compiled using Scaffold.

Proteomic map of Butte 86 total flour protein

Dupont et al., 2011. Proteome Science 9:10.

4,483 peptides

corresponded to

168 distinct protein

sequences

5 HMW-GS

22 LMW-GS

4 omega gliadins

13 gamma gliadins

23 alpha gliadins

# peptides chymo thermo tryp

gluten proteins 2785 26.1 51.9 22.0

non-gluten proteins 1698 14.0 5.1 80.9

percent of peptides

Peptides Identified by MS/MS

# spots # peptides chymo thermo tryp

HMW-GS 42 745 23.5 34.8 41.7

LMW-GS 34 814 25.6 54.4 20.0

alpha gliadins 35 691 26.5 68.5 5.1

gamma gliadins 34 405 30.9 43.5 25.7

omega gliadins 15 130 26.9 72.3 0.8

percent of peptides

Use of three enzymes was critical for MS

identification of certain gluten proteins

Maximum Average

LMW-GS 89% 48%

Alpha Gliadins 80% 54%

Gamma Gliadins 63% 44%

MS/MS Sequence Coverage

LMW-GS

89% Coverage

28 Chymo

44 Thermo

10 Tryp

BU-1

78% Coverage

3 Chymo

16 Thermo

6 Tryp

BU-6

BU-1 BU-6

Gamma Gliadins

BU-5

BU-4 Cys

Alpha Gliadins

78% Coverage

11 Chymo

18 Thermo

0 Tryp

BU-4

80% Coverage

6 Chymo

29 Thermo

3 Tryp

BU-12

contain celiac epitopes •

• • • • • • • •

do not contain celiac epitopes •

• • • •

•

Alpha gliadins with celiac epitopes were distinguished

• Many 2-DE spots contain more than one protein

• Multiple 2-DE spots may be identified as the same

protein

The flour proteome has multiple layers

of complexity

- Charge trains due to sample extraction or 2-DE

- Post-translational modifications (glycosylation,

proteolytic processing)

Need to consider the sum of all spots with the same ID to determine if

the protein responds to a treatment.

Uncovering the response of the grain to

the growth environment

Effects of post-anthesis fertilizer on the flour

proteome were studied first.

The nutritional status of the plant influences how the

wheat grain responds to temperature and drought.

• Total flour proteins from each resulting flour sample

were analyzed in triplicate by 2-DE.

• Progenesis software was used to detect spots, match

spots between gels, normalize and quantify spot volumes.

• Butte 86 plants were grown in triplicate at 24/17oC with

and without 20-20-20 NPK fertilizer.

• Of 373 protein spots detected, 51 spots increased and

104 spots decreased.

• When volumes of all spots identified as the same protein

sequence were summed, 54 unique proteins showed

responses to fertilizer.

Results

Effects of fertilizer on gluten proteins

Omega-5 161%

Omega 1,2 151%

Cys-type 148%

Omega 1,2 117%

By9 54%

Ax2* 40%

Dx5 39%

Bx7 29%

Dy10 19%

HMW-GS

Omega gliadins

Total flour protein

Most omega gliadins and HMW-GS increased with fertilizer

Alpha-gliadins

6 alpha gliadins

LMW-GS

Gamma-gliadins

No celiac epitopes

Celiac epitopes

Total flour protein

LMW-GS and alpha gliadins

showed variable responses

to fertilizer

SHIP

METSRV

METSCIP

increase

decrease

no change

Omega gliadins 144%

HMW-GS 33%

LMW-GS 15%

Alpha gliadins 31%

Gamma gliadins NC

Gliadin/Glutenin

HMW-GS/LMW-GS

Effects of fertilizer on gluten proteins

Altenbach et al. Proteome Science 9:46, 2011.

0.61 - 0.95

1.0 - 1.3

Chain-terminators omega gliadins only

Effects of fertilizer on non-gluten proteins

Total flour protein

37% Serpins

Amylase/protease inhibitors 57%

GSP/puroindoline

Beta-amylase

Other proteins that decreased:

Chitinase

Lipid transfer protein

Globulin-2

Thaumatin-like protein

Triosephosphate isomerase

Elongation factor EF1A

Glucose and ribitol dehydrogenase

Farinins

Purinins

• Post-anthesis fertilizer has complex effects on

the wheat flour proteome

Conclusions

• Study provides a basis for deciphering effects of

temperature and drought on the flour proteome

• Most notable changes are increases in omega

gliadins and decreases in a subset of LMW-GSs

How can we capture discoveries from

proteomics analyses?

Goal:

Establish links between specific proteins and

flour quality

Approach:

Silence the expression of genes encoding

specific proteins in transgenic plants

Omega gliadins

• Omega-5 gliadins are associated with food allergy

(wheat-dependent exercise-induced anaphylaxis)

• Show the largest response to post-anthesis fertilizer

• Consist of 2 protein types:

Omega-5 gliadins FPQQQ and QQIPQQ repeats

Omega-1,2 gliadins QQPFP repeats

Silencing of omega-5 gliadin genes in

transgenic Butte 86 plants

• Butte 86 plants were transformed with the RNAi

plasmid and homozygous plants were selected

• RNAi plasmid was constructed using a 153 bp target

sequence that matched all Butte 86 omega-5 gliadin ESTs

HMW-GS promoter HMW-GS terminator Omega-5 Omega-5 SS intron

2940 bp 2008 bp 153 bp 153 bp 146 bp

Non-transgenic

(whole grain)

Transgenic

(whole grain from T3 plants)

Effects of gene silencing on the proteome

Omega-5 gliadins

Transgenic Plants

Quality

Do omega-5 gliadins

influence flour quality?

How does the plant respond

to fertilizer in the absence of

omega-5 gliadins?

Summary

• A proteomic map of Butte 86 flour was developed in

which 93% of flour proteins were identified by MS/MS.

- Required knowledge of gluten protein genes expressed

in the cultivar under study.

- Required digestion of proteins with three separate

proteases.

• Improved MS/MS sequence coverage made it

possible to distinguish very similar gluten proteins.

- Required that significant changes in individual 2-DE

spots as well as different protein types be considered.

• Using the map, the complex effects of post-anthesis

fertilizer were determined in a single protein sample.

Summary

• Proteins that responded to fertilizer were targeted in

gene silencing experiments in transgenic plants.

• Transgenic plants will make it possible to relate

specific changes in the proteome to flour quality.

USDA-ARS Western Regional Research Center

Albany, California

Thank you!

Many 2-DE spots contain more than one protein Alpha and gamma gliadins and LMW-GS overlap in 2-D gels

These also overlap with non-gluten proteins

Multiple 2-DE spots may be identified as the

same protein

Need to consider the sum of all spots with the same ID to

determine if the protein responds to a treatment.

Glycosylation

alpha-amylase inhibitors

CM16 and CM17

Proteolytic

Processing

farinin (avenin-like b

protein)

Charge Trains

HMW-GS

LMW-GS