Mapping the Black Périgord Truffle proteome and ... · Proteomics identified 836 (6.54%)...
Transcript of Mapping the Black Périgord Truffle proteome and ... · Proteomics identified 836 (6.54%)...
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Mark BakerShoba RanganathanPeter & Kate MarshallAbidali Mohamedali
M.D. [email protected]
Mapping the Black Périgord Truffle proteomeand discovering what the proteins do?
ONE MANY ALL
gene genes genomics
mRNA transcripts transcriptomics
protein proteins proteomics
metabolite metabolites metabolomics
Q: Why was the genome of the Truffle sequenced in thefirst place?
A: The Périgord black truffle genome was sequenced tounderstand its evolutionary origins and mechanisms ofsymbiosis - revealed features of an ancestral fungallineage that diverged >450M yr ago
Answers lie in understanding truffle biology
2010
PathologyProteome Metabolome Biology
2020 2014 2020
2013 2003-now 2013 on 2013 on
2025
5
Genome
Proteomics/metabolomics important becauseunlike genome they change in time and space
Ulysses Caterpillar
Ulysses ButterflyBoth organismshave identical static genomes
BUTphenomenally different proteomes
One (1) of the ~7,466 protein-coding genesunequivocally identified in an early study
(prior to genome being published)
UNRIPE RIPE
Many proteome changes as fruiting body ripens(prior to genome being published)
OUR PROJECT AIMS
1. map baseline proteome (membrane &cytoplasmic proteins) of Australian blackPérigord truffle (T. melanosporum)
2. Compare the proteomes of other“truffles”
3. analyse and confirm which proteins canbe deployed as black Périgord truffle“biomarkers” of:
• Authenticity (aestivum, uncinatum, oregonense,
gibbosum, lyonii, borchii, magnatum, sinensis,himalayensis, etc)
• Optimal fungal freshness
• Fungal fruiting body deterioration
• Highly desirable perfume, aroma and taste(organoleptic; to savour (French))
Black Périgord Truffle Proteome
Freeze crush (in liquid N2)
Dissolve truffleproteins in buffer
Sonicate& spin
Run on 1D gelMW
250
191
97
64
51
39
28
19
14
14 gel cutsare then
digested intopeptides
derived fromeach protein
Black Périgord Truffle Proteome
Freeze crush (in liquid N2)
Dissolve truffleproteins in buffer
Sonicate& spin
Run on 1D gel
14 gel cutsare then
digested intopeptides
derived fromeach protein
MW
250
191
97
64
51
39
28
19
14
Périgord Truffle Proteome
Freeze crush (in liquid N2)
Dissolve truffleproteins in buffer
Sonicate& spin
Run on 1D gel
$1,000,000extremely
accurate scalecalled aMASS
SPECTROMETER
MW
250
191
97
64
51
39
28
19
14
14 gel cutsare thendigested
intopeptidesderived
from eachprotein
Périgord Truffle Proteome
Freeze crush (in liquid N2)
Dissolve truffleproteins in buffer
Sonicate& spin
Run on 1D gel
datainterrogated
againstrecently
publishedtruffle
genome
After in silicotryptic digestionof the proposed
proteome
MW
250
191
97
64
51
39
28
19
14
14 gel cutsare thendigested
intopeptidesderived
from eachprotein
$1,000,000extremely
accurate scalecalled aMASS
SPECTROMETER
One T. melanosporumprotein that predicted a
cell wall biosyntheticenzyme had been
previously identified
We now have identified 836T. melanosporum proteins
(6.54% of genome)
Identified volatiles matched to enzymes involved in their synthesis obtained fromT. melanosporum proteomic* or bioinformatics experiments
GSTUMT00008176001 is a 464amino acid containing enzyme
involved in truffle/bacterialgeneration of the metabolite
phenylacetaldehyde
We completed the first functional annotation of the black Périgordtruffle proteome from its 12,771 proteins
Using sequential search strategies we identified homologues for atotal of 2,587 proteins with 2,429 having functional annotations withhomologues from other fungi
Proteomics identified 836 (6.54%) non-redundant proteins of which388 of which were present in our bioinformatics studies.
The list of black Périgord truffle proteins that were functionallyannotated and confirmed by proteomic experiments in our study isby far the most comprehensive (truffle) study to date contributingover 2,587 proteins to the UniProt database of which 384 haveproteomic evidence
A high quality, aroma &perfume truffle
One Quality Biomarker Search Approach
An inferior quality, aroma, &perfume truffle
A Post-Genome Black Périgord Wish List
Complete & accurate genome
Complete & accurate transcriptome
Complete & accurate knowledge of whatevery truffle protein does• location and timing of protein expression• function of each protein• change in expression over life cycle• relationships with desirable qualities• structure of each protein
Complete & accurate metabolome• location• timing• conditions favorable to desirable qualities
Answers also lie in understanding human biology
2000-2010
PathologyProteome Interactome Phenome
2020 2030 2035
2014 2014 2014 2014
2040
28
Genome
Deliver a comprehensive map of human proteins and annotate all human
proteins in biological contexts
Provide tools allowing the scientific community to optimise experimental design
and biodiscovery, as the Human Genome Project did
Inspire the scientific community to understand, diagnose, prevent, treat and
cure human disease, leading to improved human health outcomes
The Human Proteome Project
121618192428
394041
4951
6165
117308
439
0 50 100 150 200 250 300 350 400 450 500
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Human Proteome Missing Proteins*(neXtProt June 2014)
olfactory receptors zinc finger proteins
transmembrane proteins coiled-coil domain proteins
keratin-associated proteins leucine-rich repeat proteins
cadherins homeobox proteins
other GPCRs ankyrin repeat & death-domain proteins
defensins nuclear proteins
late cornified envelope proteins potassium channel proteins
taste receptors ring finger proteins
* Missing Protein CriteriaLane et al., 2013 JPR
Odorant receptors and V1Rvomeronasal receptors have shortN-terminal extracellular domains
in contrast to V2Rs(number of genes in family shown in brackets)
Amino acid conservation inodorant receptor consensus
sequence
blue highly conservedred highly variable
Relationship between functional ORphylogenetic clades and chromosomal clusters
Number OR genes in each clade/cluster shown in parentheses
Clade and clusters joined by coloured lines with thicknessproportional to number of OR genes - therefore clades and clustersare joined by lines with different thickness(e.g., 8/13 OR genes in clade D are located in cluster 1.4)
Phylogeneticclade
Chromosomalcluster
Ectopic OR mRNAexpression
Heat map for 40 most highlyexpressed human tissue ORs
sorted by expression across alltissues
Reimann et al., G-Protein-Coupled Receptors in Intestinal Chemosensation. Cell Metabolism. 15, 4, 421–43, 2012.