期刊
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
卷 32, 期 6, 页码 1490-1497出版社
AMER CHEMICAL SOC
DOI: 10.1021/jasms.1c00070
关键词
diphtheria toxoid; vaccine quality control; proteomics; tandem mass tag multiplexing; enzyme kinetics; degradomics
资金
- Innovative Medicines Initiative 2 Joint Undertaking [N-115924]
Animal tests are currently used to confirm the efficacy and safety of toxoid vaccines, but in vitro assays may replace them if production consistency can be proven. A TMT labeling method based on a common reference allows for faster analysis time and the identification of suitable candidate peptides for quality control assays.
Currently, animal tests are being used to confirm the potency and lack of toxicity of toxoid vaccines. In a consistency approach, animal tests could be replaced if production consistency (compared to known good products) can be proven in a panel of in vitro assays. By mimicking the in vivo antigen processing in a simplified in vitro approach, it may be possible to distinguish aberrant products from good products. To demonstrate this, heat-exposed diphtheria toxoid was subjected to partial digestion by cathepsin S (an endoprotease involved in antigen processing), and the peptide formation/degradation kinetics were mapped for various heated toxoids. To overcome the limitations associated with the very large number of samples, we used common reference-based tandem mass tag (TMT) labeling. Instead of using one label per condition with direct comparison between the set of labels, we compared multiple labeled samples to a common reference (a pooled sample containing an aliquot of each condition). In this method, the number of samples is not limited by the number of unique TMT labels. This TMT multiplexing strategy allows for a 15-fold reduction of analysis time while retaining the reliability advantage of TMT labeling over label-free quantification. The formation of the most important peptides could be followed over time and compared among several conditions. The changes in enzymatic degradation kinetics of diphtheria toxoid revealed several suitable candidate peptides for use in a quality control assay that can distinguish structurally aberrant diphtheria toxoid from compliant toxoids.
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