Creating a detailed map of the wheat flour proteome: 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:a critical step in understanding the effects of environment on flour quality and immunogenic potential 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
Temperature Drought FertilizerQuality Proteome Transgenic Plants Genes
Outline• 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
Separation of flour proteins by 2-DE Gluten proteins Total Flour Proteincomprise ~80% of SDS + DTT flour protein Gluten Proteins Non-gluten Proteins Non-gluten Proteins KCl-insoluble KCl-soluble, MeOH-insoluble KCl-soluble, MeOH-soluble ~80% of total ~11% of total ~5% of total
Identification of proteins by tandem mass spectrometry (MS/MS) Proteins digested with trypsin Peptides separated and further fragmented, spectra generated Spectra matched to data generated in silico from protein databases MS/MS yields sequence information rather than just the mass of the peptides
MS/MS Identification Requires That• 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.
Proteomic maps of non-gluten proteinsKCl-soluble/MeOH-insoluble metabolic proteins structural proteins defense proteins Vensel et al. Proteomics 5: 1594-1611, 2005. a-amylase/trypsin inhibitorsKCl-soluble/MeOH-soluble defense proteins Wong et al.,Plant Cell Physiol. 45: 407-415, 2004.
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
Why??Gluten proteins have very repetitive sequences thatare rich in glutamine and proline % Gln + Pro HMW-GS & LMW-GS 43-54% alpha & gamma gliadins 49-56% omega gliadins 68-73% Proteins are not readily digested with trypsinThe major groups of gluten proteins contain manysimilar sequences and there is considerable sequenceheterogeneity among different cultivars Gluten protein sequence diversity is not adequately reflected in current databases
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 Chymotrypsin Thermolysin Trypsin• Optimize database by including sequences of gluten proteins from the cultivar under study
Analysis of ESTs from Butte 86 Alpha Gliadins 136 ESTs assembled into 19 contigs • 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 Gliadins153 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• 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 2,562,722 protein sequences
Identification of Gluten Proteins from Butte 86 by MS/MS• Proteins in individual 2-DE spots cleaved with trypsin, chymotrypsin, or thermolysin.• 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 5 HMW-GS 4,483 peptides 22 LMW-GS corresponded to 4 omega gliadins168 distinct protein 13 gamma gliadins sequences 23 alpha gliadins Dupont et al., 2011. Proteome Science 9:10.
Alpha gliadins with celiac epitopes were distinguished••••••••••••• • contain celiac epitopes • do not contain celiac epitopes
The flour proteome has multiple layers of complexity• Many 2-DE spots contain more than one protein• Multiple 2-DE spots may be identified as the same protein - Post-translational modifications (glycosylation, proteolytic processing) - Charge trains due to sample extraction or 2-DE 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 environmentThe nutritional status of the plant influences how the wheat grain responds to temperature and drought. Effects of post-anthesis fertilizer on the flour proteome were studied first.
• Butte 86 plants were grown in triplicate at 24/17oC with and without 20-20-20 NPK fertilizer.• 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.
Results• 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.
Effects of fertilizer on gluten proteins HMW-GS By9 54%Omega gliadins Ax2* 40% Omega-5 161% Dx5 39% Omega 1,2 117% Bx7 29% Omega 1,2 151% Cys-type 148% Dy10 19% Total flour protein Most omega gliadins and HMW-GS increased with fertilizer
LMW-GS SHIPLMW-GS and alpha gliadinsshowed variable responses METSRV to fertilizer METSCIP Alpha-gliadins 6 alpha gliadins Celiac epitopes No celiac epitopes Gamma-gliadins Total flour protein increase decrease no change
Effects of fertilizer on gluten proteins Omega gliadins 144% HMW-GS 33% Alpha gliadins 31% LMW-GS 15% Gamma gliadins NC Gliadin/Glutenin 1.0 - 1.3 HMW-GS/LMW-GS 0.61 - 0.95 Chain-terminators omega gliadins only Altenbach et al. Proteome Science 9:46, 2011.
Effects of fertilizer on non-gluten proteins Serpins 37% Amylase/protease inhibitors 57% Other proteins that decreased: Farinins Purinins Chitinase Globulin-2 Lipid transfer protein Thaumatin-like protein Total flour protein Beta-amylase Glucose and ribitol dehydrogenase Triosephosphate isomerase Elongation factor EF1A GSP/puroindoline
Conclusions• Post-anthesis fertilizer has complex effects on the wheat flour proteome• Most notable changes are increases in omega gliadins and decreases in a subset of LMW-GSs• Study provides a basis for deciphering effects of temperature and drought on the flour proteome
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• 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• Omega-5 gliadins are associated with food allergy (wheat-dependent exercise-induced anaphylaxis)
Silencing of omega-5 gliadin genes in transgenic Butte 86 plants• RNAi plasmid was constructed using a 153 bp target sequence that matched all Butte 86 omega-5 gliadin ESTs• Butte 86 plants were transformed with the RNAi plasmid and homozygous plants were selected HMW-GS promoter Omega-5 SS intron Omega-5 HMW-GS terminator 2940 bp 153 bp 146 bp 153 bp 2008 bp
Effects of gene silencing on the proteomeOmega-5 gliadins Non-transgenic Transgenic (whole grain) (whole grain from T3 plants)
How does the plant respond to fertilizer in the absence of omega-5 gliadins? Do omega-5 gliadinsinfluence flour quality? Quality Transgenic Plants
Summary• A proteomic map of Butte 86 flour was developed in which 93% of flour proteins were identified by MS/MS.• Improved MS/MS sequence coverage made it possible to distinguish very similar gluten proteins. - Required digestion of proteins with three separate proteases. - Required knowledge of gluten protein genes expressed in the cultivar under study.
Summary• Using the map, the complex effects of post-anthesis fertilizer were determined in a single protein sample. - Required that significant changes in individual 2-DE spots as well as different protein types be considered.• 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 Charge Trains HMW-GS LMW-GS Proteolytic Processing farinin (avenin-like b protein)Glycosylationalpha-amylase inhibitorsCM16 and CM17 Need to consider the sum of all spots with the same ID to determine if the protein responds to a treatment.