title: Comparison of two FFPE preparation methods using label-free shotgun proteomics: Application to tissues of diverticulitis patients.
abstract: Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic biomarkers. However, the critical step in this field is the ability to obtain an efficient and repeatable extraction using the limited quantities of material available for research in hospital biobanks. This work describes the evaluation of the peptide/protein extraction using FFPE sections treated by the following two methods before shotgun proteomic analysis: a commercial solution (FFPE-FASP) (filter aided sample preparation) and an antigen retrieval-derived protocol (On Slice AR). Their efficiencies and repeatabilities are compared using data-independent differential quantitative label-free analysis. FFPE-FASP was shown to be globally better both qualitatively and quantitatively than On Slice AR. FFPE-FASP was tested on several samples, and differential analysis was used to compare the tissues of diverticulitis patients (healthy and inflammatory tissues). In this differential proteomic analysis using retrospective clinical FFPE material, FFPE-FASP was reproducible and provided a high number of confident protein identifications, highlighting potential protein biomarkers.
  
  
  CONCLUSIONS
  In clinical proteomics, FFPE is an important resource for retrospective analysis and for the discovery of biomarkers. The challenge for FFPE shotgun proteomic analysis is preparation by an efficient and reproducible protocol, which includes protein extraction and digestion. In this study, we analyzed two different methods and evaluated their repeatabilities and efficiencies. We illustrated the reproducibility of the most efficient method, FFPE-FASP, by a pilot study on diverticulitis tissue and on FFPE samples amount accessible in hospital biobanks. These data showed that FFPE is suitable for use in clinical proteomics, especially when the FFPE-FASP method is combined with label-free shotgun proteomics as described in the workflow presented in this work.
authors.affiliation: Gastroenterology Department, GIGA-R, Liège University Hospital CHU, ULg, Liège, Belgium.
authors.forename: Florence
authors.initials: F
authors.lastname: Quesada-Calvo
authors.affiliation: Department of Preclinical and Biomedical Sciences, Mammalian Cell Culture Laboratory, GIGA-R, ULg, Liège, Belgium.
authors.forename: Virginie
authors.initials: V
authors.lastname: Bertrand
authors.affiliation: Chemistry Department, Laboratory of Mass Spectrometry, GIGA-R, CART, ULg, Liège, Belgium.
authors.forename: Rémi
authors.initials: R
authors.lastname: Longuespée
authors.affiliation: Biobanque universitaire, Liège University Hospital CHU, Liège, Belgium.
authors.forename: Agnès
authors.initials: A
authors.lastname: Delga
authors.affiliation: Chemistry Department, Laboratory of Mass Spectrometry, GIGA-R, CART, ULg, Liège, Belgium.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Mazzucchelli
authors.affiliation: Chemistry Department, Laboratory of Mass Spectrometry, GIGA-R, CART, ULg, Liège, Belgium.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Smargiasso
authors.affiliation: GIGA Proteomic Facility, ULg, Liège, Belgium.
authors.forename: Dominique
authors.initials: D
authors.lastname: Baiwir
authors.affiliation: Department of Preclinical and Biomedical Sciences, Laboratory of Cytology and Histology, GIGA-R, ULg, Liège, Belgium.
authors.forename: Philippe
authors.initials: P
authors.lastname: Delvenne
authors.affiliation: Department of Clinical Sciences, Department of Rheumatology, Liège University Hospital CHU, Liège, Belgium.
authors.forename: Michel
authors.initials: M
authors.lastname: Malaise
authors.affiliation: Department of Preclinical and Biomedical Sciences, Mammalian Cell Culture Laboratory, GIGA-R, ULg, Liège, Belgium.
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: De Pauw-Gillet
authors.affiliation: Chemistry Department, Laboratory of Mass Spectrometry, GIGA-R, CART, ULg, Liège, Belgium.
authors.forename: Edwin
authors.initials: E
authors.lastname: De Pauw
authors.affiliation: Gastroenterology Department, GIGA-R, Liège University Hospital CHU, ULg, Liège, Belgium.
authors.forename: Edouard
authors.initials: E
authors.lastname: Louis
authors.affiliation: Gastroenterology Department, GIGA-R, Liège University Hospital CHU, ULg, Liège, Belgium. Electronic address: marie-alice.meuwis@ulg.ac.be.
authors.forename: Marie-Alice
authors.initials: MA
authors.lastname: Meuwis
mesh_terms: D015415:Biomarkers / Q000032:analysis / Q000737:chemistry; D004238:Diverticulitis / Q000378:metabolism* / Q000473:pathology; D005260:Female; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D008297:Male; D016612:Paraffin Embedding; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*
chemical_list: D015415:Biomarkers; D010455:Peptides; D005557:Formaldehyde
keywords: Antigen retrieval; Data independent; FFPE; Filter aided sample preparation (FASP); Label-free; Shotgun proteomics