Each aliquot of the first five aliquots of bead-captured target was then incubated with 50 ml of a TNT-PNA probe solution comprising 1 nmol of the TNT-PNA probe complementary to the target on the beads. Similarly, the sixth aliquot was then incubated with 100 ml of a second TNT-PNA probe solution comprising 2 nmol of the TNT-PNA probe complementary to the target but with the alternative TNT tag on the probe. Hybridization of all the aliquots was carried out at room temperature for two hours. After hybridization, the probe solution was removed from the beads and the beads were washed three times with ice-cold 70 mM aqueous ammonium citrate solution. After the wash step, the sixth aliquot was resuspended in 100 ml of ammonium citrate. Aliquots of the sixth aliquot were then pipetted into the first six aliquots: 20, 10, 4, 2, and 1 ml, respectively. The mixtures The TNT tags, outlined by the red broken line, are modular comprising different functional components that correspond to individual synthetic components in the automated synthesis of these reagents. Each TNT is composed of two parts. The first part is a tag fragment, drawn in red, that comprises a charge-carrying group, the piperazine moiety outlined by the black broken box, and an alanine mass modifier group, outlined by the green broken box. The tag fragment is linked to the second part of the tag, which is a mass normalization group, drawn in blue, that ensures that each tag in a pair of tags shares the same overall mass and atomic composition. Note that the tags shown above are not strictly isobaric due to an issue with the synthesis discussed in the text. The mass normalization group is essentially uncharged and comprises a second alanine mass modifier group to adjust the overall mass of the tag and a proline group that is part of the electrospray cleavable linker. The ESI-cleavable linker is outlined by another black broken box and comprises an aspartic acid proline linkage. Part (ii) of Figure 2a Tables 1 and 2. were then mixed thoroughly. The hybridized TNT-PNA probes were then denatured from their captured target by incubating the beads at 858C for 20 min in 50:50 water:methanol with 1% formic acid. The elution solutions were then analysed by direct injection ESI-MS/MS.