research questions regulation of eukaryotic signal transduction: focussing on protein...

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Research questions Regulation of eukaryotic signal transduction: focussing on protein phosphorylation and protein-protein interactions (in the context of cancer cells invasion and metastasis) Genome-scale protein expression profiling of tumour tissue to discover novel drug target candidates New biomarkers and protein-based assays New approaches to proteomics and phosphoproteomics data analysis (in collaboration with Math Sci.) Methodology Applications of high-resolution hybrid mass spectrometry for quantitative genome- scale studies of protein abundance and post-translational modifications Cell-based models of specific pathways involved in metastasis: mutagenesis, overexpression, stable lines Structure/function studies in the context of protein phosphorylation and protein-

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CD74 overexpressed in metastatic TNBC: potential biomarker? IHC staining of CD74 in 19 TNBC specimens CD74 and Scribble spectral counts by LC-MS/MS IHC staining of CD74 in TNBC Metodieva et al. 2013, Neoplasia

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Page 1: Research questions Regulation of eukaryotic signal transduction: focussing on protein phosphorylation and protein-protein interactions (in the context

Research questions

Regulation of eukaryotic signal transduction: focussing on protein phosphorylation and protein-protein interactions (in the context of cancer cells invasion and metastasis)

Genome-scale protein expression profiling of tumour tissue to discover novel drug target candidates

New biomarkers and protein-based assays

New approaches to proteomics and phosphoproteomics data analysis (in collaboration with Math Sci.)

Methodology

Applications of high-resolution hybrid mass spectrometry for quantitative genome-scale studies of protein abundance and post-translational modifications

Cell-based models of specific pathways involved in metastasis: mutagenesis, overexpression, stable lines

Structure/function studies in the context of protein phosphorylation and protein-protein interactions

Page 2: Research questions Regulation of eukaryotic signal transduction: focussing on protein phosphorylation and protein-protein interactions (in the context

Trypsindigestion

SDS-PAGE or membrane isolationNano-LC-MS/MSa

b c

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R² = 0.931513752706484

Spectral count

Norm

alize

d AQ

UA

1 10 100 10001

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10000R² = 0.974789037975781

Spectral counts of replicate 1

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High-definition tumor proteomics using the LTQ/Orbitrap Velos

25,000+ peptide fragmentation spectra in one LC/MS run that takes 90 min. 2000+ proteins can be identified and quantified.

Page 3: Research questions Regulation of eukaryotic signal transduction: focussing on protein phosphorylation and protein-protein interactions (in the context

CD74 overexpressed in metastatic TNBC: potential biomarker?C

D74

IHC

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Node-negative Node-positive0

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10 p-value = 0.04

IHC staining of CD74 in 19 TNBC specimens CD74 and Scribble spectral counts by LC-MS/MS

IHC staining of CD74 in TNBC

Metodieva et al. 2013, Neoplasia

Page 4: Research questions Regulation of eukaryotic signal transduction: focussing on protein phosphorylation and protein-protein interactions (in the context

Quantitative phosphoproteomics and interaction analysis to elucidate mechanisms contributing to metastasis in the context of TNBC.

1. Generate stable lines that express CD74 under the control of highly-regulated inducible promoter

2. Label cells with stable isotopes to allow quantitative analysis of global protein phosphorylation.

3. Identify phosphorylation “hot spots”: protein phosphorylation sites that respond to CD74 overexpression.

Clone 1 Clone 2

Tetracycline - + - +

CD74

We use SILAC (stable isotope labelling by aminoacids in culture). We label both the arginines and the lysines to allow comprehensive phosphopeptide quantitation.

CD74 has been implicated in several kinase-regulated pathways. Therefore we want to identify not only changes in total protein abundance but also phosphorylation site changes in response to CD74 overexpression. For this we use phospho-affinity approaches and quantitative MS.

Page 5: Research questions Regulation of eukaryotic signal transduction: focussing on protein phosphorylation and protein-protein interactions (in the context

-2 -1.5 -1 -0.5 0 0.5 1 1.5 20

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LOG2(Ratio H/L Normalized)

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omed

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1: MAESPCSPSGQQPPSPPSPDEIPANVK2: MAESPCSPSGQQPPSPPSPDEIPANVK3: AFAAVPTSHPPEDAPAQPPTPGPAASPEQISFR4: QSPASPPPIGGGAPVR

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TetO_SILAC_MEMBR_Phos_1_120716200848 #6764 RT: 49.18 AV: 1 NL: 8.55E3T: FTMS + p NSI Full ms [400.00-2000.00]

988.5 989.0 989.5 990.0 990.5 991.0 991.5 992.0m/z

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989.08

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989.75988.75

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991.42991.08

992.10990.75

991.76 992.46988.96

990.42992.20

989.20

MAESPCSPSGQQPPSPPSPDEIPANVK

Light

Heavy

TetO_SILAC_MEMBR_Phos_1_120716200848 #7780 RT: 55.02 AV: 1 NL: 1.30E4T: FTMS + p NSI Full ms [400.00-2000.00]

1140 1141 1142 1143 1144 1145m/z

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1145.211142.21 1144.88

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AFAAVPTSHPPEDAPAQPPTPGPAASPEQISFR

AS1306S1309

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Scribble serine phosphorylation hot spots affected by CD74 overexpression in MCF7 and HEK293 cells

Normalized H/L ratios for detected Scribble phosphopeptides

High-resolution MS scans

p-value=0.00068

Metodieva et al. 2013. Neoplasia

Page 6: Research questions Regulation of eukaryotic signal transduction: focussing on protein phosphorylation and protein-protein interactions (in the context

Metodieva et al. 2013. Neoplasia

CD74-dependent deregulation of Scribble - confocal imaging

Page 7: Research questions Regulation of eukaryotic signal transduction: focussing on protein phosphorylation and protein-protein interactions (in the context

ScribbleBeta-Pix

GIT2GIT1

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T-test difference

GFP-Scribble co-IP/MS assay. IPs with anti-GFP and non-specific Ab as a negative control were performed on aliquots of lysate obtained from transfected HEK293 cells and analysed in triplicate by nano-LC-MS/MS. The label-free intensities for all proteins were log-transformed and specific interactors identified by the volcano plot approach . Scribble and the known interactors Beta-Pix, Git1 and Git2 are shown on the plot. Other significant proteins are shown with black symbols.

Protein-protein interaction analysis by GFP co-IP and quantitative LC-MS/MS

Page 8: Research questions Regulation of eukaryotic signal transduction: focussing on protein phosphorylation and protein-protein interactions (in the context

External

Rick Bucala, Yale University, USA

Lin Leng, Yale University, USA

David Stone, University of Illinois at Chicago, USA

Louise Aldridge, Griffith University, Australia

Roland Croner, University of Erlangen, Germany

Christina Greenwood, The Helen Rollason Heal Cancer Care Laboratory, Anglia Ruskin University, UK

Khalid Al-Janabi, Histopathology Department, Broomfield Hospital, UK

Collaborations

Internal

Berthold Lausen

Nelson Fernandez

Elena Klenova

Phil Reeves