Behaviour of TMT labelled peptides in solid phase extraction, liquid chromatography, electrospray ionisation, and data-dependent acquisition

In bottom-up proteomics, quantification with isobaric labelling involves derivatising peptides with reagents that are isobaric when intact but upon fragmentation produce reporter ions of different masses. Peptides from different samples are labelled with different reagent isotopomers, and the reporter ion intensities serve as a readout for peptide abundance in different samples, enabling sample multiplexing. Increased chromatographic retention of labelled peptides has been described and considered in models predicting peptide retention. However, how isobaric labelling affects peptide behaviour in other steps of the analytical workflow remains scarcely studied. In this work, we applied isobaric tandem mass tag (TMT) labelling on synthetic peptides to study how TMT labelling affects the peptides’ retention and yield in solid phase extraction, retention in liquid chromatography, and signal intensity. In addition, we monitored how closely reporter ion intensity reflects precursor ion intensity across chromatographic peaks. In solid phase extraction, TMT labelling caused a slight increase in retention without being detrimental to yield. In liquid chromatography, our results confirm those of earlier publications, identifying a retention increase, the magnitude of which is correlated with peptide hydrophilicity. We detected a near-consistent decrease of signal intensity in TMT-labelled peptides that may imply decreased ionisation efficiency but is likely mitigated by increased analyte amount that comes with multiplexing. Finally, we observed that although reporter-to-precursor ion ratios are mostly stable, for some peptides they vary enough to significantly affect quantification. Our results show that while TMT labelling affects peptide behaviour in analytics, changes to the analytical workflow are mostly not required.