Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping

Journal Article · Sat Dec 01 04:00:00 EST 1990 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.87.24.9848· OSTI ID:5017007

Suckau, D; Schneider, K; Przybylski, M ^[1]; Kohl, J; Karwath, G; Bitter-Suermann, D ^[2]; Casaretto, M ^[3]

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1. Univ. Konstanz (West Germany)
2. Medizinische Hochschule (West Germany)
3. Deutsches Wollforschungsinstitut Aachen (West Germany)

Sequences of antigenic determinants were identified by limited proteolysis of peptide antigens bound to an immobilized monoclonal antibody and direct molecular weight determination of the monoclonal antibody-bound peptide fragments by {sup 252}Cf plasma desorption mass spectrometry. The epitope peptides to the monoclonal antibody h453 were isolated from immobilized antigen-antibody complexes by partial trypsin digestion. A synthetic eicosapeptide comprised of the C-terminal sequence of the human complement component polypeptide des-Arg{sup 77}-C3a as well as guinea pig des-Arg{sup 78}-C3a was used as an antigen. Conditions were developed under which trypsin specifically degraded the antigens without inactivation of the immobilized antibody. After proteolysis, epitope peptides were dissociated from the antibody with 4M MgCl{sub 2}. The antigenic peptides were purified by HPLC and identified by {sup 252}Cf plasma desorption mass spectrometry. The epitope recognized by h453 resides on the C-terminal tryptic peptides of human and guinea pig C3a. As an estimation of accuracy this method is able to provide, trypsin digestion of immune complexes caused cleavage of the antigen within a distance of two amino acid residues upstream from the epitope.

• OSTI ID: 5017007
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (United States) Vol. 87:24; ISSN 0027-8424; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACTINIDE ISOTOPES
ACTINIDE NUCLEI
ALPHA DECAY RADIOISOTOPES
ANTIBODIES
ANTIGEN-ANTIBODY REACTIONS
ANTIGENS
CALIFORNIUM 252
CALIFORNIUM ISOTOPES
CHEMICAL REACTIONS
DECOMPOSITION
ENZYMATIC HYDROLYSIS
ENZYMES
EVEN-EVEN NUCLEI
HEAVY NUCLEI
HYDROLASES
HYDROLYSIS
IMMUNE REACTIONS
ISOTOPES
LYSIS
MASS SPECTROSCOPY
MOLECULAR WEIGHT
MONOCLONAL ANTIBODIES
NUCLEI
PEPTIDE HYDROLASES
RADIOISOTOPES
SERINE PROTEINASES
SOLVOLYSIS
SPECTROSCOPY
SPONTANEOUS FISSION RADIOISOTOPES
TRYPSIN
YEARS LIVING RADIOISOTOPES