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, 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

Abstract

Background: Recently, it was demonstrated that proteins can be translated from alternative open reading frames (altORFs), increasing the size of the actual proteome. Top-down mass spectrometry-based proteomics allows the identification of intact proteins containing post-translational modifications (PTMs) as well as truncated forms translated from reference ORFs or altORFs.

Methods: Top-down tissue microproteomics was applied on benign, tumor and necrotic-fibrotic regions of serous ovarian cancer biopsies, identifying proteins exhibiting region-specific cellular localization and PTMs. The regions of interest (ROIs) were determined by MALDI mass spectrometry imaging and spatial segmentation.

Findings: Analysis with a customized protein sequence database containing reference and alternative proteins (altprots) identified 15 altprots, including alternative G protein nucleolar 1 (AltGNL1) found in the tumor, and translated from an altORF nested within the GNL1 canonical coding sequence. Co-expression of GNL1 and altGNL1 was validated by transfection in HEK293 and HeLa cells with an expression plasmid containing a GNL1-FLAG(V5) construct. Western blot and immunofluorescence experiments confirmed constitutive co-expression of altGNL1-V5 with GNL1-FLAG.

Conclusions: Taken together, our approach provides means to evaluate protein changes in the case of serous ovarian cancer, allowing the detection of potential markers that have never been considered.

Keywords: Alternative proteins; Biomarkers; Hidden proteome; Microproteomics; Ovarian cancer; Top-down.

Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1
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).
Fig. 2
Fig. 2
Systems biology analysis. Global network identification of the proteins present in benign (a), necrotic-fibrotic tumor (b) and tumor regions (c).
Fig. 3
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.
Fig. 4
Fig. 4
Precursor and HCD fragmentation scan of Alternative Guanine Nucleotide-binding Protein-like 1 (AltGNL1).
Fig. 5
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 10 μm.

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