Beta Alanine -Aspartic Acid -Proline -Alanine -Piperazin-1-ylacetic acid -13 C 3 , 15 N-Alanine between the tags. Thus, the overall masses of the tags are the same but the tag fragments have different masses, which are equalized by the mass of the mass normalization portion. These tags are designed so that on analysis by collision-induced dissociation (CID), the tag fragment is released to give rise to a uniquely resolvable ion. Thus, this pair of tags allows a pair of PNA probes to be distinguished by MS/MS analysis. Each tag is linked to a PNA oligonucleotide probe by a second linker, comprising aspartic acid and proline that is easily cleaved by CID (37) . As shown in Figure 2 , the aspartic acid/proline linker is used to cleave the tags from their oligonucleotides during electrospray ionization of the tagged oligonucleotides. The expected structures and mass-to-charge ratios of the cleaved parent tag ions generated by dissociation of the aspartic acid/proline linkage are shown in part (ii) of figure 2a and b (37) . Similarly, the expected structures and mass-to-charge ratios of the tag fragment ions and the structures of the neutral mass normalizer fragments, based on the predictions of the 'mobile proton' model of peptide fragmentation (38, 39) , are shown in part (iii) of Figure 2a and b. The use of FMOC peptide synthesis chemistry to synthesize the TNTs combined with FMOC PNA chemistry to synthesize the oligonucleotide probe sequence means the same resin can be used to synthesize both portions of the tagged probe in a single total synthesis approach. For the probes discussed here, the TNT portion of the probe was synthesized in a commercial peptide synthesizer using standard peptide synthesis resin cartridges. The cartridge was then opened and the resin within was loaded into a cartridge suitable for a commercial oligonucleotide synthesizer that supports PNA synthesis (Expedite DNA/PNA synthesizer), allowing the completion of the synthesis of the probe. The completed probe was then cleaved from the resin and deprotected in one step (TFA/cresol) and then purified. With this approach, only one purification step is required resulting in better yields of finished probe than multi-stage processes. Figure 3 illustrates the ease with which a substantial number of tags can be synthesized from a small set of mass modifier components: nine tags, shown in Figure 3b can be made from three mass modifiers, which are the three commercially available isotopes of alanine shown in Figure 3a . In fact, the number of tags that can be synthesized increases as the square of the number of mass modifier components, e.g. there are at least five isotopes of alanine with different masses that are commercially available which would actually allow the synthesis of 25 tags using the design presented here.