Upload
cimmyt-int
View
738
Download
3
Tags:
Embed Size (px)
DESCRIPTION
International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012
Citation preview
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