title: In situ proteomic analysis by MALDI imaging identifies ubiquitin and thymosin-β4 as markers of malignant intraductal pancreatic mucinous neoplasms.
abstract: OBJECTIVE
  Intraductal pancreatic mucinous neoplasms (IPMN) are precancerous cystic lesions. The aim was to investigate the in situ IPMN proteome using MALDI (Matrix-Assisted Laser Desorption/Ionisation) imaging and to characterize biomarkers associated with the grade of dysplasia.
  
  
  METHODS
  Frozen human Branch duct -IPMN sections were selected according to dysplasia and proteomic analyses were performed by MALDI imaging to obtain mass spectra distribution. The most discriminating peaks were identified using tissue extraction and nanoLC-ESI-MS/MS. Identified peaks were validated in independent series of IPMN by immunochemistry on surgical specimens (tissue-microarrays (TMA), n = 45) and endoscopic ultrasound fine-needle aspiration (EUS FNA) samples (n = 25).
  
  
  RESULTS
  BD-IPMN samples with low (n = 10) and high (n = 10) grades of dysplasia were analyzed. Differential spectra of proteins were found in the two groups with significantly different intensities (n = 15). The two peaks (intense in high grade IPMN) (m/z 8565 and 4747) were characterized as the monomeric ubiquitin (Mascot score = 319.22) and an acetylated fragment of thymosin-β4 (2-42) (Omssa score = 1.37 E-9). Validation on TMA and EUS FNA samples confirmed that ubiquitin was overexpressed in high grade dysplasia (p = 0.04 and p = 0.0004). Thymosin-β4 expression was confirmed on TMA by immunohistochemistry on high grade IPMN (p = 0.011).
  
  
  CONCLUSIONS
  Ubiquitin and thymosin-β4 are overexpressed in IPMN with high grade dysplasia. Positive immunochemical staining on EUS-FNA material is a major argument in support of preventive resection.
authors.affiliation: Pancreatology Department, Beaujon Hospital, AP-HP, Clichy, Paris-Diderot University, France; Inserm U773-CRB3, Paris-Diderot University, Paris, France. Electronic address: vinciane.rebours@bjn.aphp.fr.
authors.forename: Vinciane
authors.initials: V
authors.lastname: Rebours
authors.affiliation: Inserm U773-CRB3, Paris-Diderot University, Paris, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Le Faouder
authors.affiliation: Inserm U773-CRB3, Paris-Diderot University, Paris, France.
authors.forename: Samira
authors.initials: S
authors.lastname: Laouirem
authors.affiliation: Inserm U773-CRB3, Paris-Diderot University, Paris, France.
authors.forename: Mounya
authors.initials: M
authors.lastname: Mebarki
authors.affiliation: Inserm U773-CRB3, Paris-Diderot University, Paris, France; Pathology Department, Beaujon Hospital, AP-HP, Clichy, Paris-Diderot University, France.
authors.forename: Miguel
authors.initials: M
authors.lastname: Albuquerque
authors.affiliation: Mass Spectrometry Facility, Jacques Monod Institute, UMR7592, Paris-Diderot University - CNRS, Paris, France; Molecular and Cellular Pathology Program, Jacques Monod Institute, UMR7592, Paris-Diderot University - CNRS, Paris, France.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Camadro
authors.affiliation: Mass Spectrometry Facility, Jacques Monod Institute, UMR7592, Paris-Diderot University - CNRS, Paris, France.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Léger
authors.affiliation: Pancreatology Department, Beaujon Hospital, AP-HP, Clichy, Paris-Diderot University, France; Inserm U773-CRB3, Paris-Diderot University, Paris, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Ruszniewski
authors.affiliation: Pancreatology Department, Beaujon Hospital, AP-HP, Clichy, Paris-Diderot University, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Lévy
authors.affiliation: Inserm U773-CRB3, Paris-Diderot University, Paris, France; Pathology Department, Beaujon Hospital, AP-HP, Clichy, Paris-Diderot University, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Paradis
authors.affiliation: Inserm U773-CRB3, Paris-Diderot University, Paris, France; Pathology Department, Beaujon Hospital, AP-HP, Clichy, Paris-Diderot University, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Bedossa
authors.affiliation: Inserm U773-CRB3, Paris-Diderot University, Paris, France; Pathology Department, Bichat Hospital, AP-HP, Paris-Diderot University, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Couvelard
mesh_terms: D000368:Aged; D000595:Amino Acid Sequence; D014408:Biomarkers, Tumor / Q000032:analysis; D005260:Female; D006801:Humans; D006868:Hydrolysis; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D018297:Neoplasms, Cystic, Mucinous, and Serous / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D010183:Pancreatic Ducts / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013947:Thymosin / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D000099201:Thymosin beta(4)
chemical_list: D014408:Biomarkers, Tumor; D013947:Thymosin; D014357:Trypsin; D025801:Ubiquitin; D000099201:Thymosin beta(4)
keywords: Biomarker hunting; IPMN; Imaging mass spectrometry; MALDI; Pancreatic precancerous lesions; Proteomics; Thymosin-β4; Ubiquitin