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Characterization of disulfide bridges pattern of recombinant human interleukin 12 fusion protein p40 subunit and identification and quantification of cysteinylated free cysteine 252

Rapid Commun. Mass Spectrom.
January 17, 2024

Researchers at Merck Serono show evidence of cysteinylation on Cys252 of recombinant human p40 subunit of interleukin 12 (IL-12). This was reported in 1996. However, no paper detailing this concept has been published yet. This paper reports the quantification of Cys252 cysteinylation as well as the full disulfide bridges assignment by nonreducing peptide mapping using mass spectrometry (MS) detection. Nonreducing peptide mapping was applied for disulfide bridges assignment. This study presents an ad hoc method in which applying a neutral pH in the presence of an alkylating agent allowed to mitigate the formation of artifacts such as reshuffled disulfide bridges and permitted the detection of free cysteine. Ultra-high-performance liquid chromatography–MS analysis was performed on a Waters quadrupole time-of-flight Xevo G2-XS mass spectrometer acquiring data in MSE mode. MS data were processed using Genedata Expressionist.