title: Topologies of amyloidogenic proteins in Congo red-positive sliced sections of formalin-fixed paraffin embedded tissues by MALDI-MS imaging coupled with on-tissue tryptic digestion.
abstract: OBJECTIVE
  Matrix-assisted laser desorption time-of flight ionization (MALDI)-imaging MS (IMS) with MSMS analysis using on-tissue tryptic digests is a powerful tool for identification of disease-related proteins in formalin-fixed paraffin-embedded (FFPE) tissue sections. We applied this novel IMS technique, not only to identify tryptic peptides of deposited amyloidogenic proteins but also to clarify topologies of these proteins in amyloidosis tissue sections.
  
  
  METHODS
  Sequence determinations of tryptic peptides derived from amyloidogenic proteins were performed using MALDI-MSMS analysis directly from Congo red positive regions in tissue sections with/without procedure for retrieval of epitopes before on-tissue digestion.
  
  
  RESULTS
  Tryptic peptides, m/z=1073.5 and 1924.3 were identified with the sequences, from 48th to 56th and 1st to 19th positions of Ig lambda V-III region, respectively. Other peptides, m/z=1365.5 and 1523.5 were with the sequences, from 22nd to 34th and 36th to 48th positions of TTR, respectively. Heat-map images of all four tryptic peptides were overlapped with Congo red positive regions. Immunohistochemistry of FFPE tissue sections was confirmed to only react with anti-λ chain antibody in a case of AL-type amyloidosis or anti-TTR antibody in two cases of TTR-type amyloidosis.
  
  
  CONCLUSIONS
  IMS with MSMS analysis using on-tissue tryptic digestion enables us not only to identify amyloidogenic molecule in a sliced tissue section but also to play a complementary role with the conventional pathological examination.
authors.affiliation: Department of Clinical and Laboratory Medicine, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki-city 569-8668, Osaka, Japan. Electronic address: nakanisi@poh.osaka-med.ac.jp.
authors.forename: Toyofumi
authors.initials: T
authors.lastname: Nakanishi
authors.forename: Minako
authors.initials: M
authors.lastname: Ito
authors.forename: Takashi
authors.initials: T
authors.lastname: Nirasawa
authors.forename: Motomu
authors.initials: M
authors.lastname: Tsuji
authors.forename: Takayuki
authors.initials: T
authors.lastname: Takubo
mesh_terms: D000368:Aged; D000595:Amino Acid Sequence; D028227:Amyloid Neuropathies, Familial / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D058227:Amyloidogenic Proteins / Q000032:analysis / Q000737:chemistry*; D000686:Amyloidosis / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D003224:Congo Red; D005260:Female; D005557:Formaldehyde; D006801:Humans; D000075363:Immunoglobulin Light-chain Amyloidosis; D007146:Immunoglobulin lambda-Chains / Q000032:analysis / Q000737:chemistry*; D008297:Male; D008867:Microtomy; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D011228:Prealbumin / Q000032:analysis / Q000737:chemistry*; D059748:Proteolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
chemical_list: D058227:Amyloidogenic Proteins; D007146:Immunoglobulin lambda-Chains; D010446:Peptide Fragments; D011228:Prealbumin; D005557:Formaldehyde; D003224:Congo Red; D014357:Trypsin
keywords: Amyloidosis; Immunoglobulin lambda; MALDI-MS imaging; On-tissue trypsin digestion; Transthyretin