One issue that emerged from the experiments presented here was a loss of 13 C isotope from the carboxylic acid of alanine when the alanine isotope was present at the C-terminus of the peptide TNT, i.e. in TNT2 shown in Table 1 . This loss is consistent across every TNT-PNA synthesized with TNT2 and may be an effect of the resin used, which relied on a 4-HydroxyMethyl-Phenoxy Acetic acid (HMPA) linker to the carboxylic acid group of the first amino acid to allow the peptide to be cleaved from the resin at the end of the synthesis. Other resins will be tested in future to avoid the loss of isotope. This has meant that the TNTs synthesized were not completely isobaric as shown in Figure 2 . The mass of the singly charged parent tag ion of TNT1 is 388.2 while that of TNT2 is 387.2. For the MS/MS analysis of these tags, both tags could still be selected simultaneously by the first quadrupole of the Q-TOF instrument, as the mass range that is gated is actually about 3 daltons for the default setting of The TNT approach is similar in principle to other mass tagging techniques and enjoys the same features as other approaches, such as ease of multiplexing and the ability to design tag masses to suit applications, with some additional advantages. TNT tags can be made chemically identical, even sharing the same mass as long as the tag fragments are different, so they can act as more precise reciprocal internal standards, which leads to more accurate quantification and the same behaviour in analytical separations, hybridizations and labelling reactions thus avoiding 'dye effects' that plague fluorescent methods (40, 41) . The use of an MS/MS-based detection method allows TNTs to be selected from background noise thus improving signal to noise ratios. This allows untagged material to be ignored, greatly improving data quality.