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.

中文翻译：

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重组蛋白破伤风类毒素重链片段 C 中半胱氨酸修饰的研究。

对于结合 HIV-1 融合肽疫苗的开发，重组破伤风类毒素重链片段 C (rTTHC) 被用作载体蛋白以增强肽免疫原性。了解 rTTHC 的特性是肽偶联之前的第一步。在其纯化过程中对大肠杆菌表达的 rTTHC 进行了全面的质谱 (MS) 表征。完整质量以及肽图分析发现存在三种半胱氨酸修饰形式：谷胱甘肽化、三硫键修饰和二硫键改组，与疏水相互作用色谱 (HIC) 纯化步骤中的三峰分布相关。这些多种氧化形式的共存表明活性硫醇在 rTTHC 材料中经历了氧化还原反应。