1. 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

2. Trypsin digestion SDS-PAGE or membrane isolation Nano-LC-MS/MS a b c 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.

3. 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

4. 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.

5. 1: MAESPCSPSGQQPPSPPSPDEIPANVK 2: MAESPCSPSGQQPPSPPSPDEIPANVK 3: AFAAVPTSHPPEDAPAQPPTPGPAASPEQISFR 4: QSPASPPPIGGGAPVR 1 2 3 4 B C MAESPCSPSGQQPPSPPSPDEIPANVK Light Heavy Light Heavy AFAAVPTSHPPEDAPAQPPTPGPAASPEQISFR A S 1306 S 1309 S 1348 S 1448 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

6. CD74-dependent deregulation of Scribble - confocal imaging

7. 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

8. 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