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:
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Creating a detailed map of the wheat flour proteome: a critical step in understanding the effects of environment on flour quality and immunogenic potential
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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