In this article the synthesis, characterization and application of ESI-cleavable TNT peptide-PNA conjugates to the quantitative detection of target DNA sequences by ESI-MS/MS has been described. The conjugates were prepared by first synthesizing the TNT tag peptide sequence in a peptide synthesizer, after which the peptide synthesis resin was transferred to a column compatible with a DNA synthesizer in which PNA can be prepared. The PNA sequence was extended directly from the peptide TNT. The use of PNA has several advantages for this application: (i) the oligonucleotide and the peptide TNT are generated in a single synthesis on the same resin, which means only a single purification step is required after the synthesis is completed; (ii) PNA is approximately 15% lower in mass than a corresponding DNA sequence, which Figure 8 . The relationship between the expected and measured ratio of mixtures of captured target sequences probed with 12-mer TNT-PNA probes. Experiments to quantify targets by external calibration where the PNA probes had identical sequences but different TNTs are shown with blue crosses and the blue regression line while experiments to quantify targets by internal calibration, i.e. using one sequence as a reference to quantify a different sequence are shown as red squares and the red regression line. enhances mass spectrometric sensitivity; (iii) PNA has enhanced binding affinity for its target compared with a corresponding DNA sequence, so a shorter probe can be used to achieve a corresponding level of specificity; (iv) PNA can hybridize under low salt conditions, which is more favourable for ESI analysis of samples as ESI-MS is susceptible to signal suppression by high salt concentrations.