Spatial XL-MS : Spatially-resolved cross link mass spectrometry guided by Mass spectrometry

Support : ANR
: Julia Chamot Rooke (Pasteur Paris)

Spatial XL-MS is based on the a spatially resolved highly sensitive analytical platform (SpatialXomics) which can generate valuable proteomic, metabolomic information while avoiding ion suppression guided by MALSI MSI and targets quantitative image reconstruction. To achieve this challenging goal, we propose a new spatially resolved analytical technique where images are reconstructed based on data coming from identification/quantification analysis. This will be achieved by rostering the tissue section using liquid micro-extraction system and performing identification and relative quantification LC ESI MS to analyse the extracts from all pixels. Based on our results, we believe that our spatially resolved analytical technique will be extremely useful to reveal fundamental roles of proteins in physio-pathological processes by getting access to the deep proteome and the ghost proteome. Thus, the overarching goal of the SpatialXOmics project is to develop a new experimental and computational strategy to generate MS images based on identification/quantification data for proteins highlighting spatial abundance of identified proteins. Spatial XL-MS is now integrating cross-link technology to identify in tissue protein-protein interaction which open the door of structural analyses in tissues.

Spatially resolved XL-MS workflow from tissue regions and example of an identified intramolecular interaction between STXBP1 and PPPRC


  • Lemaire R, Desmons A, Day R, Salzet M, Fournier I (2007) Direct analysis and MALDI imaging on formalin fixed paraffin embedded tissue sections (FFPE). J. Proteome Res. 6(4):1295-305
  • Djidja MC, Francese S, Loadman PM, Sutton CW, Scriven P, Claude E, Snel MF, Franck J, Salzet M, Clench MR Detergent addition to tryptic digests and ion mobility separation prior to MS/MS improves peptide yield and protein identification for in situ proteomic investigation of frozen and formalin-fixed paraffin-embedded adenocarcinoma tissue sections. Proteomics. (2009) 9(10):2750-63
  • Franck J, Arafah K, Barnes A, Wistzorski M, Salzet M, Fournier I Improving tissue preparation for MALDI-MSI: Part 1, (2009) 81(19):8193-202
  • Franck J, El Ayed M, Wisztorski M, Salzet M, Fournier I On Tissue N-terminal peptide derivatizations for enhancing protein identification in MALDI-MSI strategies. Anal. Chem (2009) 81(20):8305-17
  • Bonnel D, Longuespee R, Franck J, Roudbaraki M, Gosset P, Day R, Salzet M, Fournier I Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer. Anal Bioanal Chem. (2011) 401(1):149-65
  • Frank J, Longuespée R, Wisztorski M, Van Remoortere A, Van Zeijl R, McDonnell L, Salzet M, Fournier I MALDI mass spectrometry imaging of proteins exceeding 30 000 Da. Med Sci Monit. (2010 ) 16(9):BR293-9
  • Mériaux C, Frank J, Wisztorski M, Salzet M, Fournier I ionic liquid matrices as matrix for mass spectrometry imaging of lipids. J. Proteomics (2010) 73(6):1204-18
  • Van Remoortere A, van Zeijl RJ, van den Oever N, Franck J, Longuespée R, Wisztorski M, Salzet M, Deelder AM, Fournier I, McDonnell LA. MALDI imaging and profiling MS of higher mass proteins from tissue. J Am Soc Mass Spectrom. (2010) 21(11):1922-9
  • Bruand J, Sistla S, Mériaux C, Dorrestein PC, Gaasterland T, Ghassemian M, Wisztorski M, Fournier I, Salzet M, Macagno E, Bafna V Automated Querying and Identification of Novel Peptides using MALDI Mass Spectrometric Imaging. J Proteome Res. (2011) 10(4):1915-28
  • Bruand JA, Sistla T, Wisztorski M, Meriaux C, Becker M, Salzet, M, Fournier I, Macagno E, Bafna V  AMASS : Algorithm for MSI Analysis by Semi-supervised Segmentation, J. Proteome Res. (2011)10(10):4734-43
  • Quanico J, Franck J, Dauly C, Strupat K, Dupuy J, Day R, Salzet M, Fournier, I, Wisztorski M Development of liquid microjunction extraction strategy for improving protein identification from tissue sections. Journal of Proteomics 2013, 79, 200-218
  • Wisztorski M, Fatou, B.; Franck, J.; Desmons, A.; Farre, I.; Leblanc, E.; Fournier, I.; Salzet, M., Microproteomics by liquid extraction surface analysis: Application to FFPE tissue to study the fimbria region of tubo-ovarian cancer. Proteomics Clinical Applications 2013, 7, (3-4), 234-240.
  • Quanico J, Franck J, Gimeno JP, Sabbagh R, Salzet M, Day R, Fournier I Parafilm-assisted microdissection: a sampling method for mass spectrometry-based identification of differentially expressed prostate cancer protein biomarkers. Chem Commun (Camb). (2015) 51(22):4564-7. (Journal Cover)
  • Quanico J, Franck J, Salzet M, Fournier I On-tissue Direct Monitoring of Global Hydrogen/Deuterium Exchange by MALDI Mass Spectrometry: Tissue Deuterium Exchange Mass Spectrometry (TDXMS). Mol Cell Proteomics. (2016) 15(10):3321-3330
  • Quanico J, Franck J, cardon T, Leblanc E, Wisztorski M, Salzet M, Fournier I NanoLC-MS Coupling of Liquid Microjunction Microextraction for On-Tissue Proteomic Analysis. Biochim Biophys Acta. (2016) 9. pii: S1570-9639(16)30229-1
  • Quanico J, Franck J, Wisztorski M, Salzet M, Fournier I Integrated Mass Spectrometry Imaging and Omics Workflows on the Same Tissue Section Using Parafilm-Assisted Microdissection. Biochim Biophys Acta. (2017) pii: S0304-4165(17)
  • Quanico J, Franck J, Wisztorski M, Salzet M, Fournier I Combined MALDI Mass Spectrometry Imaging and Parafilm-Assisted Microdissection-Based LC-MS/MS Workflows in the Study of the Brain. Methods Biol. (2017); 1598:269-283
  • Wisztorski M, Desmons A, Quanico J, Fatou B, Gimeno JP, Franck J, Salzet M, Fournier I Spatially-resolved protein surface microsampling from tissue sections using liquid extraction surface analysis. Proteomics (2016) 16(11-12):1622-32
  • Wisztorski M, Quanico J, Franck J, Fatou B, Salzet M, Fournier I Droplet-Based Liquid Extraction for Spatially-Resolved Microproteomics Analysis of Tissue Sections. Methods Mol Biol.( 2017);1618:49-63
  • Delcourt V, Franck J, Quanico J, Gimeno JP, Wisztorski M, Raffo Romero A, Kobeissy F, Roucou X, Salzet, M, Fournier, I (2017) Top-down microproteomics bridged to MALDI MS imaging reveals the molecular physiome of brain regions. Molecular & cellular proteomics : Molecular and Cellular Protemics 2018 17(2):357-372

IMHOTHEP Single Cell Tumor Heterogeneity by Organoids Large Scale Proteomics

Support : AVIESAN


Since response to treatment is highly dependent on cell phenotypes, tumor heterogeneity (TH) is a clear hindrance to therapy development. Due to epigenetic modifications, TH is not only spatial but temporal as well. To improve cancer treatment, it is therefore critical to tackle the molecular modifications associated to the spatio-temporal evolution of TH. Patient tissues-derived organoids represent interesting and relevant models to study TH. Thus, the molecular characterization of each cell within organoids is an important step towards TH characterization. The goal of the IMHOTEP project is to develop a robust workflow to obtain the characterization of TH from organoids through single cell transcriptomic analysis and combined these data to electrophysiology measurements and single cell proteomics data. The project principle is to perform the single cell transcriptomic analysis of the organoids, use the identified markers to sort out cells using FACS, and further select single cells for patch-clamp and proteomics. Through patient-derived organoids, this strategy will be applied to glioblastoma, which prognostic is pejorative and is known to display a high TH. The project will be organized in 4 work packages. WP1 will be dedicated to the development of glioblastoma organoids and WP2 to single cell proteomics. In WP3 we will integrate all the developments into a robust and reproducible workflow which will be applied in WP4. This study will be performed on a prospective cohort of 10 patients and will shed light on gliobastoma TH by combining the single cell data from transcriptomics, proteomics and electrophysiology measurements.

Ghost : proteogenomic associated to MALDI MSI for targeting alternative proteins involved in oncological processes

Support : Cancéropole Nord Ouest
: Xavier Roucou, Andreas Tholey


The recent advances of high throughput sequencing in the field of genomics and mass spectrometry (MS) based proteomics, it has been shown that traditional computational genome annotation algorithms have underestimated the number of open reading frames (ORFs). Recent evidence has shown that small ORFs (smORFs) are present in RNAs including non-coding RNAs. Recently, PRISM has shown that many microproteins translated from smORFs can be detected in cancer including ovarian cancer and glioma. Even if their existence has been widely proven, their biological functions remain to be elucidated. Thus GHOST project will respond to questions focusing on the role of microproteins and therefore their involvement in the glioma. Novel potential therapeutic targets can be identified and may not lead to tumor resistance or secondary effects of treatment. Additionally, large scale proteomics analysis combining microproteins and canonical proteins will provide more insights about patient profiles in response to specific treatments. Using SpatialXOmics associated to Top-down technology but also Cross link mass spectrometry (XL-MS) allow to investigate the function of these Ghost proteins.


  • Vanderperre B, Lucier JF, Tremblay G, Motard J, Venderperre S, Wisztorski M, Salzet M, Boisvert FM, Roucou X Direct identification of alternative open reading frames translation products in human. PLoS One 2013, 8(8), e70698
  • Mouilleron H, Delcourt V, Roucou X Death of a dogma: eukaryotic mRNAs can code for more than one protein Nucleic Acids Res. 2016 Jan 8;44(1):14-23
  • Delcourt V, Franck J, Leblanc E, Narducci F, Robin YM, Gimeno JP, Quanico J, Wisztorski M, Kobeissy F, Jacques JF, Roucou X, Salzet M, Fournier I Combined Mass Spectrometry Imaging and Top-down Microproteomics Reveals Evidence of a Hidden Proteome in Ovarian Cancer. EBioMedicine. (2017) 21:55-64
  • Delcourt V, Staskevicius A, Salzet M, Fournier I, Roucou X (2018) Small Proteins Encoded by Unannotated ORFs are Rising Stars of the Proteome, Confirming Shortcomings in Genome Annotations and Current Vision of an mRNA. Proteomics (2017) 18(10):e1700058
  • Delcourt V, Brunelle M, Roy AV, Jacques JF, Salzet M, Fournier I, Roucou X. First evidence that a protein coded by a short ORF, not by the annotated CDS is the main gene product. Mol Cell Proteomics. 2018 Dec;17(12):2402-2411
  • Samandi S, Roy AV, Delcourt V, Lucier JF, Gagnon J, Beaudoin MC, Vanderperre B, Breton MA, Motard J, Jacques JF, Brunelle M, Gagnon-Arsenault I, Fournier I, Ouangraoua A, Hunting DJ, Cohen AA, Landry CR, Scott MS, Roucou X Deep transcriptome annotation enables the discovery and functional characterization of cryptic small proteins. Elife. 2017 Oct 30;6. pii:
  • Delcourt V, Staskevicius A, Salzet M, Fournier I, Roucou X. Small Proteins Encoded by Unannotated ORFs are Rising Stars of the Proteome, Confirming Shortcomings in Genome Annotations and Current Vision of an mRNA. Proteomics. 2018 May;18(10):e1700058.
  • Brunet MA, Brunelle M, Lucier JF, Delcourt V, Levesque M, Grenier F, Samandi S, Leblanc S, Aguilar JD, Dufour P, Jacques JF, Fournier I, Ouangraoua A, Scott MS, Boisvert FM, Roucou X OpenProt: a more comprehensive guide to explore eukaryotic coding potential and proteomes. Nucleic Acids Res. 2019 Jan 8;47(D1):D403-D410
  • Cardon T, Salzet M, Franck J, Fournier I Nuclei of HeLa cells interactomes unravel a network of Ghost-proteins involved in proteins’ translation. Biochim Biophys Acta Gen Subj. 2019 May 23. pii: S0304-4165(19)30130-8
  • Cardon T, Hervé F, Delcourt V, Roucou X, Salzet M, Franck J, Fournier I Optimized Sample Preparation Workflow for Improved Identification of Ghost ProteinsAnal Chem. 2020 Jan 7;92(1):1122-1129
  • Murgoci AN,Cardon T, Aboulouard S, Duhamel M, Fournier I, Cizkova D, Salzet M Reference and Ghost proteins identification in Rat C6 glioma extracellular vesicles. IScience (In press)
  • Cardon T, Franck J, Damato M, Maffia M, Vergara D, Fournier I, Salzet M Alternative Proteins are Functional Regulators in Cell Reprogramming by PKA Activation. Nucleic Acid Research (In press)

BAT-MASS new kit for multiplexing and quantifying transcriptome, proteome, glycome, metabolome by targeting MALD-MSI

Support : SATT Nord
: Nucleosyn


To date, many strategies can be used as tools for clinical diagnosis and these can be classified according to their mode of applications. The most popular ones are ELISA, immunohistochemistry (IHC), immuno PCR, fluorescence in situ hybridization (FISH) and the Multiplex ligation-dependent probe amplification (MLPA), to name some. Recently, the use of MS has emerged as a powerful technique for diagnosis. Indeed, the indirect detection of biomarkers has been developed such as the Multiplexed ion beam imaging (MIBI) and the imaging mass cytometry which was recently described as histology topography cytometry analysis toolbox (histoCAT). All these strategies present the ability to perform molecular images of proteins in a multiplexed mode. Nevertheless, these techniques are based from what we have previously developed with the Tag-mass technology in targeted MALDI MSI and in ELISA-based assay, named ISA-MS. This technology has been patented in 2005 and worldwide delivery. Even if all the mentioned methods are able to localize biomolecules in the multiplexed mode, the development of a universal tool to detect both nucleic acids and proteins remains challenging and has to be elucidated. BAT-Mass project aims to synthesize a nucleotide-linked kit, MS-detectable Tag allowing the in situ or in vitro detection and quantify of poteome, glycome, transcriptome, proteome as well as drugs.


  • Lemaire R, Stauber J, Wisztorski M, Van Camp C, Desmons A, Deschamps M, Proess G, Rudlof I, Woods AS, Day R, Salzet M, Fournier I (2007) Tag-Mass: Specific Molecular Imaging of Transcriptome and Proteome by Mass Spectrometry Based on Photocleavable Tag, Proteome Res. 6(6):2057-67
  • Stauber J, El Ayed M, Wisztorski M, Day R, Fournier I, Salzet M PCR and immunoassay – MALDI mass spectrometry using Tag-Mass technology: New tools to break down quantification limits and multiplexes. Anal. Chem (2009) 81(22):9512-21
  • Gagnon H, Franck J, Wisztorski M, Day R, Fournier I, Salzet M Targeted mass spectrometry Imaging: Specific Targeting Mass Spectrometry imaging technologies from history to perspective. Progress in Histopathology and Cytochemistry (2012) 47(3):133-74


  • Fournier I, Salzet M, ISA-MS et PCR-MS, US divisionnaire de la WO 2007/000669 A2, PCT, 2009
  • Fournier I, Salzet M, Antibodies with Conjugates with Linkers Cleavable by Photodissociation, US divisionnaire de la WO 2007/000669 A2, PCT, 2009
  • Fournier I, Lemaire R, Dechamps M, Tabet JC, Salzet M, Use of Conjugates with Linkers Cleavable by Photodissociation of Fragmentation for Mass, WO 2007/000669 A2, 2007, Delivred in EU & US

Fimbria : Molecular histology guided Spatially resolved large scale microproteomic coupled to HR MALDI MSI in tubal serous carcinoma and precancerous lesions

Supports : INCA (PHRC), Cancéropole Nord Ouest, H2020
: Comprehensive Care Center Oscar Lambret, ESGO


Whereas the ovarian cancer is only at the 7th position in women’s cancer, it is by far the most lethal (only 30-40% survival rate at 5 years). Amongst the different classes, the High Grade Serous Carcinomas (HGSC) is the most represented (>75%) and within this class, 10% of the patients are mutated for BRCA1/2 or presents a Lynch syndrome. They showed an increased risk of 20-40% to present this HGSC from 30-35 years old (versus 1.4% in the global population). In such women, without efficient detection method, the recommended action is the prophylactic adnexectomies (ovary and tubal ablations). Histopathological studies on the excised pieces showed that these HGSC are taking their origin not in the ovary but in the fimbrial part of the fallopian tube from precursor lesions called STICs (Serous tubal intraepithelial carcinoma). In this project, prophylactic adnexectomies, tissues were prepared following the See-FIM protocol (Serial Extended Examination of the FIMbria) and ImunoHistoChemistry (IHC) against p53 and Ki-67 allowed the pathologist to precisely explore the tubal extremities. Lesion of interest (“p53 lesion”, STIC, STOUT, and HGSC) were analyze by HR MALDI MSI coupled to spatially resolved tissue micro-proteomic guided by molecular histology in FFPE tissue.


  • Lemaire S, Aït-Menguellet J, Stauber JP, Lucot P, Collinet MO, Farine D, Vinatier R, Day P, Ducoroy M, Salzet M, Fournier I (2007) Biomarker Discovery and Validation by Profiling and Specific Imaging by Mass Spectrometry : Reg-Alpha Fragment is a Biomarker of Ovary Cancer. J. Proteome Res. 6(11):4127-34
  • Vergara D, Simeone P, Toraldo D, Del Boccio P, Vergaro V, Lepratti S, Piragostino D, Tinelli A, De Domenico S, Urbani A, Salzet M, Santino A, Maffia M Resveratrol downregulates Akt/GSK and ERK signalling pathways in OVCAR-3 ovarian cancer cells ; Mol Biosyst. (2012),8(4):1078-87
  • Lonsguespée R, Boyon C, Kerdraon O, Desmons A, Vinatier D, Fournier I, Day R, Salzet M C-terminal fragment of the immunoproteasome PA28S (Reg Alpha) an early diagnosis and tumor relapse biomarkers: Evidences by Mass Spectrometry Profiling. Histochem Cell Biol. (2012) 138(1):141-54
  • El Ayed M, Bonnel D, Longuespée R, Castelier C, Franck J, Vergara D, Desmons A, Tasiemski A, Kenani A, Vinatier D, Day R, Fournier I, Salzet M MALDI imaging mass spectrometry in ovarian cancer for tracking, identifying, and validating biomarkers. Med Sci Monit. (2010) 16(8):BR233-45
  • El Ayed M, Bonnel D, Longuespée R, Castellier C, Franck J, Vergara D, Desmons A, Tasiemski A, Kenani A, Vinatier D, Day R, Fournier I, Salzet M MALDI Imaging Mass Spectrometry in Ovarian Cancer for Tracking, Identifying & Validating Biomarkers Case Reports and Clinical Practice Review (2010), 16(8): 233-245
  • Wisztorski M, Fatou B, Franck J, Desmons A, Farre I, Leblanc E, Fournier I, Salzet M Microproteomics by liquid extraction surface analysis: Application to FFPE tissue to study the fimbria region of tubo-ovarian cancer. Proteomics Clinical Applications 2013, 7, (3-4), 234-240
  • Longuespee R, Gagnon H, Boyon C, Strupat K, Dauly C, Kerdraon O, Ighodaro A, Desmons A, Dupuis J, Wisztorski M, Vinatier D, Fournier I, Day R, Salzet M Proteomic analyses of serous and endometrioid epithelial ovarian cancers - Cases studies - Molecular insights of a possible histological etiology of serous ovarian cancer. Proteomics Clinical Applications 2013, 7, (5-6), 337-354
  • Hanssen S, Collinet P, Leblanc E, Salzet M, Vinatier D Immunological analogies between ovarian cancer and pregnancy. Journal De Gynecologie Obstetrique et Biologie De La Reproduction 2013, 42, (3), 217-226
  • Longuespée R, Couture F, Levesque C, Kwiatkowka A, Desjardins R, Gagnon S, Vergara D, Fournier I, Salzet M, Day R PACE4 is a Promising Therapeutic Target for Ovarian Cancer. Translational Oncology (2014) S1936-5233(14)00043-6
  • Giudetti A, De Domenico S, Ragusa A, Lunetti P, Gaballo A, Franck J, Simone P, Nicolardi G, De Nuccio F, Santino A, Capobianco L, Lanuti P, Fournier I, Salzet M, Maffia M, Vergara D A specific lipid metabolic profile is associated with the epithelial mesenchymal transition program. BBA - Molecular and Cell Biology of Lipids (2019);1864(3):344-357.)

SentiRad : New approaches to sentinel nodes in ovarian Cancer

Support : INCA (PHRC)
: Comprehensive Care Center Oscar Lambret, ESGO, Chieti University (Pr. S. Albertini)


Since the last 15 years, ovarian cancer pathology (OVC) has been investigated transversally in PRISM by MALDI-MSI coupled to spatially resolved tissue micro-proteomic. This apaproach was used on ovarian cancer tissues for hunting specific biomarkers and for research on cancer specific markers in OVC and in their sentinel nodes. Novel set of biomarkers involve in immune response inhibition and tolerance have been discovered and their etiology investigated. Knowledge of the origin of the different OVC permits well-fitted prophylactic measures. Moreover, the description of molecular specificity of the proteins of each subtype will permit definition of early biomarkers, useful for early diagnosis. Thus, molecular insights will identify therapeutic targets and clinical trials should no longer aggregate the various ovarian types. Two markers reg-Alpha and Trop-2 are now under multicenter investigation as novel diagnosis markers for OVC. Based on these results, therapeutic target has been investigated and the prohormone convertases PACE4 seems a promising one (See the Therapeutic Axis). OVC project is now in two directions i.e. validating MALDI MS molecular signature on large cohort for clinical diagnosis and prognosis in conjunction with the pathologist and macrophages M1 phenotype reactivation in tumor though PC1/3 inhibition (MacBeth Project).