title: Antigen digestion on the target plate of MALDI-TOF MS after isolation using an immunoaffinity membrane.
abstract: A combination of methods is required to achieve separation of intact proteins and subsequently perform structure analysis to examine their unstable or external structures. The aim of this study was to develop a method of structure analysis in intact proteins after purification. Transferrin from human plasma was trapped by membrane-immobilized anti-transferrin antibody, which was produced by non-denaturing two-dimensional electrophoresis (2-DE), and transferred to a polyvinylidene fluoride (PVDF) membrane and stained with Ponceau S. The antigen transferrin was eluted by rinsing the membrane with trifluoroacetic acid (TFA) or aspartic acid. In addition, a method was established by which the purified human transferrin was enzymatically digested on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) target plate. Thus, after purification of the human transferrin antigen from tens of microlitres of human plasma using an immunoaffinity membrane, transferrin polypeptide fragments were obtained on the plate following digestion with pepsin in the presence of 0.1% TFA or endoproteinase Lys-C or Lys-C/trypsin with 0.001% sodium dodecyl sulphate (SDS). The results indicated that the combined methods of isolation using an immunoaffinity membrane and enzymatic digestion on a MALDI-TOF MS plate could be applied to the purification and microanalysis of antigens. This approach would be particularly applicable to the analysis of the primary structure and the less stable and highly accessible regions of antigens from limited sample volumes.
authors.affiliation: Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Matsuyama, Japan. shimazaki.yoji.my@ehime-u.ac.jp
authors.forename: Youji
authors.initials: Y
authors.lastname: Shimazaki
authors.forename: Yuri
authors.initials: Y
authors.lastname: Nishimura
authors.forename: Masaki
authors.initials: M
authors.lastname: Saito
mesh_terms: D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000737:chemistry* / Q000276:immunology*; D001224:Aspartic Acid / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011145:Polyvinyls / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014168:Transferrin / Q000276:immunology; D014269:Trifluoroacetic Acid / Q000737:chemistry; D005465:Fluorocarbon Polymers
chemical_list: D000906:Antibodies; D000941:Antigens; D001224:Aspartic Acid; D010455:Peptides; D011145:Polyvinyls; D014168:Transferrin; D014269:Trifluoroacetic Acid; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers