Common Reference-Based Tandem Mass Tag Multiplexing for the Relative Quantification of Peptides: Design and Application to Degradome Analysis of Diphtheria Toxoid

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.