Open AccessInvestigation of Cysteine Modifications in Recombinant Protein Tetanus Toxoid Heavy Chain Fragment C
Author(s) -
Cindy X. Cai,
Nicole A. Schneck,
Taryn J. Cozine,
Vera B. Ivleva,
Daniel Ragheb,
Deepika Gollapudi,
Aakash Patel,
Nathan Barefoot,
Daniel B. Gowetski,
Q. Paula Lei
Publication year - 2021
Publication title -
journal of the american society for mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.961
H-Index - 127
eISSN - 1879-1123
pISSN - 1044-0305
DOI - 10.1021/jasms.1c00075
Subject(s) - chemistry , cysteine , peptide , toxoid , recombinant dna , combinatorial chemistry , mass spectrometry , biochemistry , chromatography , antigen , enzyme , biology , gene , genetics , immunization
For conjugated HIV-1 fusion peptide vaccine development, recombinant Tetanus toxoid heavy chain fragment C (rTTHC) was applied as a carrier protein to boost peptide immunogenicity. Understanding the characteristics of rTTHC is the first step prior to the peptide conjugation. A comprehensive mass spectrometry (MS) characterization was performed on E. coli expressed rTTHC during its purification process. Intact mass along with peptide mapping analysis discovered the existence of three cysteine modification forms: glutathionylation, trisulfide bond modification, and disulfide bond shuffling, in correlation to a three-peak profile during a hydrophobic interaction chromatography (HIC) purification step. Coexistence of these multiple oxidative forms indicated that the active thiols underwent redox reaction in the rTTHC material. Identity confirmation of the rTTHC carrier protein by MS analysis provided pivotal guidance to assess the purification step and helped ensure that vaccine development could proceed.