1

Mark BakerShoba RanganathanPeter & Kate MarshallAbidali Mohamedali

M.D. Islammark.baker@mq.edu.au

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

Economics ($)

mics“the study of all of life”

Transcriptomics

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

Nature March 2010

Again, why Proteomics?

Because the proteome changes in time and space

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

T. melanosporumcomputer peptide

database

T. melanosporumpeptides

Separate truffle peptides

One T. melanosporumprotein that predicted a

cell wall biosyntheticenzyme had been

previously identified

We now have identified 836T. melanosporum proteins

(6.54% of genome)

Top 5 KEGG Pathways for bioinformatics and proteomics analysis

Pictorial representation of GO distributions for the 836 T. melanosporum proteins

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

Human Chr7 Missing Proteins* (neXtProt June 2014)

121618192428

394041

4951

6165

117308

439

0 50 100 150 200 250 300 350 400 450 500

1

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

S M E L L

To Why Good Food Smells Good

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.