Primarily known for its function in the electron transport chain, cytochrome c (Cyt c) also plays a critical role in the initiation of mitochondrially induced apoptosis through specific interactions with cardiolipin (CL), a negatively charged phospholipid found in the inner mitochondrial membrane. In this work, we study the conformational dynamics of Cyt c in the presence of CL and phosphatidylcholine (PC) phospholipids also present in the mitochondrial membrane to better understand how these interactions might drive transformation to the peroxidase-active protein. Using ion mobility mass spectrometry and millisecond hydrogen−deuterium exchange mass spectrometry, we demonstrate heterogeneity in the lipid-bound ensemble, with zwitterionic (PC) phospholipids inducing destabilization of residues necessary for peroxidase coordination, and increased dynamics on the proximal face of the heme binding pocket. In contrast to what might be expected from classical models for CL-driven Cyt c peroxidase activation, interactions with CL are shown to rigidify heme coordination. To reconcile this observation with the well-supported view that CL is linked to peroxidase activation, we propose a mechanism in which CL stabilizes the conformational transition between the peroxidase-active and inactive forms.
The antigens for acellular pertussis vaccines are made up of protein components that are purified directly from Bordetella pertussis (B. pertussis) bacterial fermentation. As such, there are additional B. pertussis toxins that must be monitored as residuals during process optimization. This paper describes a liquid chromatography mass spectrometry (LC-MS) method for simultaneous analysis of residual protein toxins adenylate cyclase toxin (ACT) and dermonecrotic toxin (DNT), as well as a small molecule glycopeptide, tracheal cytotoxin (TCT) in a Pertussis toxin vaccine antigen. A targeted LC-MS technique called multiple reaction monitoring (MRM) is used for quantitation of ACT and TCT, which have established limits in drug product formulations. However, DNT is currently monitored in an animal test, which does not have an established quantitative threshold. New approaches for DNT testing are discussed, including a novel standard based on concatenated quantitation sequences for ACT and DNT. Collectively, the method represents a “3-in-1” analytical simplification for monitoring process-related residuals during development of B. pertussis vaccines.
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