Quantification of hybridized probes was also evaluated. In the first experiment, external calibration of the quantities of hybridized probes was assessed, i.e. the amount of target in one sample was probed with a TNT-PNA whose abundance was then determined by comparison with a second reference sample comprising a predefined quantity of the duplex of the same target sequence and PNA probe sequence, but with a different TNT isotope conjugated to the probe, after the hybridization. In these studies, aliquots of a synthetic biotinylated 50-mer DNA oligonucleotide target was captured onto avidinated magnetic beads and hybridized with TNT-PNA probes with identical probe sequences but different tags. The hybridized beads were then mixed in different ratios. The target, arbitrarily selected, was a fragment of a sequence from the MOMP gene from Chlamydia pneumoniae. A fixed quantity of one TNT-PNA probe complementary to one of the targets was hybridized to the captured target sequences. The aliquots of beads were then washed extensively to remove probe that had not hybridized. The captured TNT-PNA probe mixture was then eluted into 50:50 water:methanol with 1% formic acid (a solvent suitable for ESI-MS/MS analysis) by thermal denaturation. The eluted TNT-PNA and the spike were then injected directly into the Q-TOF instrument for MS/MS analysis. The ratios of the intensities of the two tags derived from the TNT-PNAs should allow the amount of the target sequences in the pooled samples to be determined. Figure 8 shows the actual correlation between the expected and measured quantities of the target sequences. The results are very similar to the experiments where TNT-PNA probes with the same sequence but different tags are simply mixed together: the measured ratio matches very closely the expected ratio. Negative controls in which the target was absent do not show significant binding of TNT-PNA conjugates to the beads so the probe binding is sequence specific. This gives a clear indication that the probes behave quantitatively in hybridization assays and that TNT-PNA probes can be used for accurate quantification.