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

Abstract

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.

Keywords: degradomics; diphtheria toxoid; enzyme kinetics; proteomics; tandem mass tag multiplexing; vaccine quality control.

Figure 1

Schematic overview of the common reference-based tandem mass tag labeling strategy. Diphtheria toxoid was exposed to various temperatures and subsequently digested by cathepsin S at 37 °C. The enzymatic reaction was stopped at various digestion time points. Aliquots of each sample were pooled to form a common reference sample. Subsequently, each sample was labeled with a unique TMT label for every temperature (the same TMT channels were used for the same exposure temperature) and a unique label for the pooled common reference sample (TMT¹¹-131C). Since each digestion sample (time point and temperature) was prepared in triplicate, there was an opportunity for extra pooled control samples, made by labeling a pooled sample containing just one triplicate. These additional controls were labeled with TMT¹¹-130N, TMT¹¹-130C, and TMT¹¹-131N, one for each triplicate. For simplicity, these are not depicted in the schematic overview (except for the MS3 spectrum). After labeling, the various heat-exposed samples were mixed with the other heat-exposed samples of the same time point and with the common reference. The mixed samples were then measured by nanoscale LC–MS, identified by MS1 and MS2, and quantified by the reporter ions generated in MS3. The reporter ions can be compared to the common reference reporter ion (TMT¹¹-131C) for every separate analysis, allowing for comparison between different runs (i.e., different time points and replicates). The peptide used for the MS3 spectra in this example is YPGLT.

Figure 2

Peptide quantification quality. (A) The identified and quantified peptides sorted by TMT reporter ion coverage, expressed as the percentage of the total number of data points (462) where the reporter was detected. The selected peptides shown in Figure 3 are marked green. (B) Example of a kinetic plot of a peptide with 50% reporter ion coverage (ILPG). (C) Example of a kinetic plot of a peptide with only 19% reporter ion coverage (VAQVIDSETADNLE). For panels B and C, up to six data points per time point at a given temperature are expected (triplicates measured twice). The relative abundance compared to the average intensity of a particular peptide over all points (the common reference) is plotted over time for panels B and C.

Figure 3

Kinetic profiles of a selection of representative peptides annotated to the diphtheria toxin crystal structure (PDB: 1DDT). The following color codes have been used: tan regions are part of the A fragment, gray regions are part of the B fragment and red is used to highlight the peptides. The relative abundance compared to the average intensity of a particular peptide over all points (the common reference) is plotted over time. Error bars represent the SD of the digestions of a diphtheria toxoid sample incubated and digested in triplicate that was measured in duplicate (i.e., six data points).

Figure 4

Base peak chromatogram of a pooled and TMT-labeled diphtheria toxoid after 32 h of cathepsin S digestion. The N-termini of the annotated peptides are TMT-labeled. The annotated peptides are those depicted in Figure 3 with increasing intensities over time.