title: A laser microdissection-based workflow for FFPE tissue microproteomics: Important considerations for small sample processing.
abstract: Proteomic methods are today widely applied to formalin-fixed paraffin-embedded (FFPE) tissue samples for several applications in research, especially in molecular pathology. To date, there is an unmet need for the analysis of small tissue samples, such as for early cancerous lesions. Indeed, no method has yet been proposed for the reproducible processing of small FFPE tissue samples to allow biomarker discovery. In this work, we tested several procedures to process laser microdissected tissue pieces bearing less than 3000 cells. Combined with appropriate settings for liquid chromatography mass spectrometry-mass spectrometry (LC-MS/MS) analysis, a citric acid antigen retrieval (CAAR)-based procedure was established, allowing to identify more than 1400 proteins from a single microdissected breast cancer tissue biopsy. This work demonstrates important considerations concerning the handling and processing of laser microdissected tissue samples of extremely limited size, in the process opening new perspectives in molecular pathology. A proof of the proposed method for biomarker discovery, with respect to these specific handling considerations, is illustrated using the differential proteomic analysis of invasive breast carcinoma of no special type and invasive lobular triple-negative breast cancer tissues. This work will be of utmost importance for early biomarker discovery or in support of matrix-assisted laser desorption/ionization (MALDI) imaging for microproteomics from small regions of interest.
authors.affiliation: Mass Spectrometry Laboratory, Systems Biology and Chemical Biology, GIGA-Research, University of Liège, Liège, Belgium; Proteopath GmbH, Trier, Germany. Electronic address: r.longuespee@molekularpatho-trier.de.
authors.forename: Rémi
authors.initials: R
authors.lastname: Longuespée
authors.affiliation: Mass Spectrometry Laboratory, Systems Biology and Chemical Biology, GIGA-Research, University of Liège, Liège, Belgium.
authors.forename: Deborah
authors.initials: D
authors.lastname: Alberts
authors.affiliation: Department of Pathology, University of Liège Hospital, Liege, Belgium; GIGA Cancer, University of Liège, Liège, Belgium.
authors.forename: Charles
authors.initials: C
authors.lastname: Pottier
authors.affiliation: Mass Spectrometry Laboratory, Systems Biology and Chemical Biology, GIGA-Research, University of Liège, Liège, Belgium.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Smargiasso
authors.affiliation: Mass Spectrometry Laboratory, Systems Biology and Chemical Biology, GIGA-Research, University of Liège, Liège, Belgium.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Mazzucchelli
authors.affiliation: GIGA Proteomic Facility, University of Liège, Liège, Belgium.
authors.forename: Dominique
authors.initials: D
authors.lastname: Baiwir
authors.affiliation: Institute of Pathology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Kriegsmann
authors.affiliation: Department of Pathology, University of Liège Hospital, Liege, Belgium; GIGA Cancer, University of Liège, Liège, Belgium.
authors.forename: Michael
authors.initials: M
authors.lastname: Herfs
authors.affiliation: Proteopath GmbH, Trier, Germany; MVZ for Histology, Cytology and Molecular Diagnostics Trier, Trier, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Kriegsmann
authors.affiliation: Department of Pathology, University of Liège Hospital, Liege, Belgium; GIGA Cancer, University of Liège, Liège, Belgium.
authors.forename: Philippe
authors.initials: P
authors.lastname: Delvenne
authors.affiliation: Mass Spectrometry Laboratory, Systems Biology and Chemical Biology, GIGA-Research, University of Liège, Liège, Belgium.
authors.forename: Edwin
authors.initials: E
authors.lastname: De Pauw
mesh_terms: D000941:Antigens / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002853:Chromatography, Liquid / Q000379:methods*; D019343:Citric Acid / Q000737:chemistry / Q000276:immunology; D005557:Formaldehyde; D006801:Humans; D007834:Lasers; D042282:Microdissection; D016612:Paraffin Embedding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics; D053719:Tandem Mass Spectrometry / Q000379:methods*; D016707:Tissue Fixation
chemical_list: D000941:Antigens; D011506:Proteins; D005557:Formaldehyde; D019343:Citric Acid
keywords: Biomarkers; Cancer; Formalin fixed paraffin embedded; Laser microdissection; Mass spectrometry; Proteomics