Combined Mass Spectrometry Imaging and Top-down Microproteomics Reveals Evidence of a Hidden Proteome in Ovarian Cancer Vivian Delcourt, Julien Franck,

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Combined Mass Spectrometry Imaging and Top-down Microproteomics Reveals Evidence of a Hidden Proteome in Ovarian Cancer Vivian Delcourt, Julien Franck, Eric Leblanc, Fabrice Narducci, Yves-Marie Robin, Jean-Pascal Gimeno, Jusal Quanico, Maxence Wisztorski, Firas Kobeissy, Jean-François Jacques, Xavier Roucou, Michel Salzet, Isabelle Fournier EBioMedicine Volume 21, Pages 55-64 (July 2017) DOI: 10.1016/j.ebiom.2017.06.001 Copyright © 2017 The Author(s) Terms and Conditions

Fig. 1 Association of MALDI-MSI and top-down microproteomics. (a) MALDI-MSI of lipids and optical image, (b) histological annotation and segmentation analysis using the Bisecting k-Means and Correlation Distance approach (left). (c) Venn diagram of the top-down gene reference products identified in the ovarian cancer tissue by the LMJ approach, the PAM approach and total, (d) Precursor and HCD fragmentation scan of Keratin 2 cytoskeletal 8 fragment 425–483 and Macrophage migration inhibitory factor (MIF). EBioMedicine 2017 21, 55-64DOI: (10.1016/j.ebiom.2017.06.001) Copyright © 2017 The Author(s) Terms and Conditions

Fig. 2 Systems biology analysis. Global network identification of the proteins present in benign (a), necrotic-fibrotic tumor (b) and tumor regions (c). EBioMedicine 2017 21, 55-64DOI: (10.1016/j.ebiom.2017.06.001) Copyright © 2017 The Author(s) Terms and Conditions

Fig. 3 GO Enrichment analysis of benign (a), necrotic/fibrotic tumor (b) and tumor region (c). (d) Global network analysis between benign and necrotic-fibrotic tumor region, between tumor and necrotic-fibrotic tumor, and between benign and tumor showed proteins involved in cell death, cell growth, keloid and muscle cell differentiation. EBioMedicine 2017 21, 55-64DOI: (10.1016/j.ebiom.2017.06.001) Copyright © 2017 The Author(s) Terms and Conditions

Fig. 4 Precursor and HCD fragmentation scan of Alternative Guanine Nucleotide-binding Protein-like 1 (AltGNL1). EBioMedicine 2017 21, 55-64DOI: (10.1016/j.ebiom.2017.06.001) Copyright © 2017 The Author(s) Terms and Conditions

Fig. 5 Validation of co-expression of reference protein GNL1 and its alternative protein AltGNL1. (a) Schematic representation of the mRNA product from GNL1 (AltGNL1) plasmid used for validation. (b) Western blot showing co-expression of GNL1 (FLAG tagged) and AltGNL1 (V5 tagged) and (c) immunofluorescence assay showing co-expression at cell level with nucleus staining (DAPI, blue) AltGNL1-V5 (green), GNL1 (red) and merge panel. White bars represent 10μm. EBioMedicine 2017 21, 55-64DOI: (10.1016/j.ebiom.2017.06.001) Copyright © 2017 The Author(s) Terms and Conditions