Download Ms. Robyn Klemptner

Comparative enrichment of
Phosphopeptides from ergosteroltreated A.thaliana leaves
Robyn Klemptner
University of Johannesburg
MSc supervisors:
Dr. L.A. Piater
Prof. I.A. Dubery
Prof. R. Meijboom
Background
BIGGEST CHALLENGE: 9 BILLION
people by 2050!!!

Food security – global importance.

Plant exposed to multiple pathogens.

Price hikes – plant diseases.

Preformed defenses.

Innate immunity = overcome pathogens.

PAMP-Triggered Immunity (PTI) + Effector-triggered
immunity (ETI).
(Lochman & Mikes, 2006 ; Godfray, 2009)
Innate immune responses









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Figure 1: A figure that clearly indicates the two mechanisms of pathogen
detection and induction of corresponding immune responses.
MAMPs/PAMPs
Preformed defenses
compromised.
Bind PRR at cell
membrane.
Signal transduction.
WRKYs.
MAMP/PAMP-triggered
immunity (M/PTI).
Effectors
Against specific host.
Suppress M/PTI.
Effector-triggered
immunity (ETI).
Recognized by
intracellular receptors.
ROS, HR, SAR.
(Klemptner et al., 2014)
Ergosterol – an “orphan” MAMP

Ergosterol = Fungal sterol, fungal cell membrane
component.

Implicated in major crop losses world wide.

Receptor/signal transduction pathway not yet elucidated.

Trigger immune response in sugar beet, grape, tomato and
tobacco plants.

Reactive oxygen species, ion fluxes, PR proteins, LTPs.
A
B
C
D
E
F
Figure 2: 3D models of various sterol compounds that have been used to
study receptor interactions in plant-pathogen interactions. A: Ergosterol; B:
Brassicasterol; C: Sitosterol; D: Stigmasterol; E: Campesterol; F: Cholesterol.
(Avrova et al., 2004; Wang , 2004; Rossard et al., 2010; Weete et al., 2010; Klemptner et al., 2014)
What we know….
 Calcium-dependent
protein kinases – Ca2+
influx.
 Phospholipase Kinase
C.
 MAPKs.
 WRKY transcription
factors.
 Phenylpropanoid
pathway – metabolites.
 H2O2 generation.
 Ergosterol perception is
specific.
Proteomics VS Genomics and
Metabolomics
Genomics
= genetic level = mRNA….
But mRNA = protein? NOT ALWAYS!
“Lost in Translation”
Proteomics
= key players in signaling.
= receptors, kinases, PR-proteins.
Metabolomics
= metabolites: jasmonates etc.
= overlapping/intersecting.
= “end products”
= pathways???
Phosphorylation
= Post-translational modification
= structural change
= functional change
Serine, Threonine and Tyrosine residues of proteins
= kinases
= signal transduction activation.
Kinases vs Phosphatases = regulation.
(Schulze, 2010)
Phosphoproteins & signal transduction
Figure 3: An overview of signal transduction pathways in defense responses in plants.
(Yang et al, 1997; Thurston et al., 2005)
Enriching phosphoproteins
Important players in signal transduction BUT occur in low
abundance! < only transiently phosphorylated!
Provide a greater knowledge of defense-related signal
transduction networks.
Methods of enrichment include:
 Affinity chromatography
 Antibody-based affinity capture
 Chemical derivatization
 Metal ion-based affinity capture
Thus, more sensitive and reliable method required =
DENDRIMERS!
Novel proteome investigation in plants since dendrimerbased enrichment techniques have yet to be applied to
plant studies.
(Meimoun et al., 2007; Iliuk et al., 2010)
Dendrimers
Figure 4: Dendrimer nanopolymers of varying generations.
(Holister et al., 2003)
Dendrimer isolation mechanism
Add dendrimer to
tryptic digest
Filter through spincolumn to isolate
dendrimer + bound
peptides
Phosphorylated
groups bind to
surface amino
groups
Cleave peptides by
acid hydrolysis
Figure 5: The fundamental dendrimer-based phosphopeptide isolation mechanism.
(Peters, 2005)
PolyMAC and PAMAM
 Dendrimers with modified terminal
groups on the surface.
 Specific affinity for phosphorylated
amino acid residues.
A
B
Figure 6A & B: The PolyMAC dendrimer and its 2 types of side-chain moieties; the traditional PAMAM dendrimer with amine
surface groups.
(Iliuk et al., 2010; Mandeville & Tajmir-Raihi, 2010)
Hypothesis
Dendrimer-based technologies
provide enhanced phosphopeptide
enrichment from A.thaliana following
ergosterol elicitation.
Objectives
1.
2.
3.
4.
5.
Elicitation of A.thaliana with
ergosterol and total protein
expression profiles.
Enrich plant phosphopeptides
using dendrimer technologies.
Compare efficiencies of PAMAM vs.
PolyMAC dendrimer enrichment
techniques.
Successful identification of
differentially expressed
phosphorylated proteins by Mass
spectrometry.
Possibly elucidate ergosterolinduced signal transduction
pathway of A. thaliana .
Methodology
PAMP treatment of A.thaliana plants
 Untreated control
 250 nM ergosterol
 EtOH control
 0, 6, 12, 24, 48, 72 hr and 7 days




Total protein extraction
Liquid N2
TCA/acetone/phenol
Ammonium acetate/meOH
precipitation
Buffers for downstream protocols
Protein concentration quantification
 Amido black assay
 BSA standards (0.625, 1.25, 2.5,
5 and 10 ug/uL)
 Samples and standards –
nitrocellulose membrane
 Absorbance at 600 nm
SDS sample buffer
 SDS-PAGE gels (1D)
 Western blotting
Urea sample buffer
 PolyMAC and PAMAM enrichment
IEF sample buffer
 Isoelectric focusing (2D)
(Granado, 1995; Lochman and Mikes, 2004; Wang et al., 2006)
Methodology
SDS-PAGE (1D)
 10 ug total/lane
 10% gel
 Fairbanks/silver staining
Western Blotting
 1° Ab
= Anti-active MAPK
= Anti-phosphoTyr
Dendrimer enrichment
 Trypsin digest
 C-18 peptide clean up
 Enrichments
= PAMAM
=PolyMAC
IEF (2D-PAGE)
 pH 3-10 and pH 4-7
 Fairbanks/silver staining
Mass spectrometry analysis
 MALDI-TOF
=DHB/CHCA
 LC-MS/MS
 Peptide sequences
 Protein ID = MASCOT
SDS-PAGE: total protein
kDA
260
M
M
140
100
70
50
40
35
~27 kDa
25
15
10
0hr
6hr
12hr
24hr
48 hr
72hr
7 days
Figure 8: SDS-PAGE separation of all protein samples. Despite there being a large number of bands that are
common to all the samples, there is a protein that shows differential expression and has an approximate size of 27
kDa.
Table 1: Protein identities following Mass Spectrometry of gel slices
Accession
Description
MW [kDa]
calc. pI
P94072
Germin-like protein subfamily 3 member
21.8
6.76
Q9ZUU4
Ribonucleoprotein At2g37220, chloroplastic
30.7
5.16
Q9FN48
Calcium sensing receptor, chloroplastic
41.3
9.39
Q05431
L-ascorbate peroxidase 1, cytosolic
27.5
6.13
O65282
20 kDa chaperonin, chloroplastic
26.8
8.88
Q9SIU8-2
Isoform 2 of Probable protein phosphatase 2C 20
30.5
6.14
Q41951
Aquaporin TIP2-1
25.0
5.64
Q0WP12-2
Isoform 2 of Thiocyanate methyltransferase 1
25.3
4.82
O24456
Guanine nucleotide-binding protein subunit beta-like protein A
35.7
7.71
Q41963
Aquaporin TIP1-2
25.8
5.06
Q8LAA6
Probable aquaporin PIP1-5
30.6
8.82
Q96291
2-Cys peroxiredoxin BAS1, chloroplastic
29.1
7.44
P42742
Proteasome subunit beta type-1
24.6
7.40
Q9LS02
Allene oxide cyclase 2, chloroplastic
27.6
7.43
P42758
Dehydrin Xero 2
20.9
9.38
O23016
Probable voltage-gated potassium channel subunit beta
36.5
7.42
P46422
Glutathione S-transferase F2
24.1
6.35
Q9SRH5
Mitochondrial outer membrane protein porin 1
29.4
8.73
Q9LHA7
Peroxidase 31
35.3
9.06
Q9ZRW8
Glutathione S-transferase U19
25.6
6.04
O04834
GTP-binding protein SAR1A
22.0
7.53
P43297
Cysteine proteinase RD21a
50.9
5.41
Q9ZTW3
Vesicle-associated membrane protein 721
24.7
8.75
P28186
Ras-related protein RABE1c
23.8
7.83
P41916
GTP-binding nuclear protein Ran-1
25.3
6.86
P41088
Chalcone--flavonone isomerase 1
26.6
5.50
Q39258
V-type proton ATPase subunit E1
26.0
6.40
P94040
Germin-like protein subfamily 3 member
21.5
9.20
O64518
Metacaspase-5
44.8
6.61
Q84W80-2
Isoform 2 of F-box/LRR-repeat protein
22.8
8.22
O81147
Proteasome subunit alpha type-6-B
27.3
6.09
Q42592
L-ascorbate peroxidase S, chloroplastic/mitochondrial
40.4
8.28
Q8LE52
Glutathione S-transferase DHAR3, chloroplastic
28.5
7.74
P43286
Aquaporin PIP2-1
30.5
8.40
P42760
Glutathione S-transferase F6
23.5
6.23
P19366
ATP synthase subunit beta, chloroplastic
53.9
5.50
A
B
pH 4 - 7
C
pH 4 - 7
D
pH 4 - 7
pH 4 - 7
Figure 9A, B, C & D: 2D-PAGE gels (11.25%) of ergosterol-treated samples following IEF,on a pH 4-7 IPG strip.
Figure A shows spots resulting from the untreated control and those in figure B show those resulting from a 0
hour ergosterol treatment. Figures C and D show spots resulting from a 6 hr and 12 hr ergosterol treatment
respectively.
Western Blotting – Anti phosphotyrosine
~40 kDa
~27 kDa
UT
0hr
6hr
12hr
24hr
48 hr
72hr
7 days
Figure 10: Autoradiography films showing Tyrosine-phosphorylated proteins following Western
blotting. The dotted yellow boxes indicate a ~27 kDa protein that exhibits a strong binding signal to
the anti-active phosphotyrosine antibody.
Western blotting – Anti active MAPK
~ 40 - 45 kDa
~ 15 - 25 kDa
UT
0hr
6hr
12hr
24hr
Figure 11: Autoradiography film showing the presence of MAPKs at 42 – 45 kDa.
MALDI-TOF mass spectrometry
 Preliminary analysis of phosphopeptide enrichment.
 DHB and CHCA matrices.
 α-casein/BSA standard + samples + calibration peptides.
 Bruker Daltonics AutoFlex at the CSIR, Biosciences.
 Nitrogen laser/ positive ion mode.
MALDI-TOF
Figure 12: MALDI-TOF spectra of phosphopeptide standard (α-casein/BSA) and PolyMAC enriched sample.
Conclusions

Preliminary MALDI analysis
indicates successful
phosphopeptide enrichment.

Anti-PhosphoTyr = specific
phosphoproteins.

~27 kDa protein across
samples = phosphorylated
protein. Confirm identity.

Ergosterol-specific proteins =
germin-like protein.

Defense and stress-related
proteins are evident =
aquaporins, LRR, calcium
binding, Ras-related protein.
(Klemptner et al., 2014)
Further studies and research
outcomes
 Final LC-MS/MS analysis = CSIR (Pretoria)/CPGR (Cape Town).
 Identify total differentially expressed proteins.
 Compare to western blots, SDS-PAGE and 2D.
 Compare enrichment of in-gel digested proteins to proteins in
solution – efficiency of dendrimer-based enrichments.
 Compare genomic, proteomic and metabolomic data.
 Dr. L. Piater, Prof. Dubery, Prof. R. Meijboom.
 Prof. A.W. Tao – Tymora Analytical/ Purdue University –
Indiana, USA.
 National Research Foundation.
 Dr. Stoyan Stoychev – CSIR Biosciences, Pretoria.
 Dr. Salome Snyman – Stellenbosch University.
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Thank you