Abstract

Analysis of 24 TMT 10-plex batches revealed an inflation in missing values and reduced inter-batch accuracy as multiple TMT batches are integrated. Our data also highlights the incidence of false positives exemplified by Y chromosome peptides being detected in female channels. The Y chromosome peptides were then used to quantify the effects of coisolation and reporter ion interference on TMT quantification and to propose an experimental design that would minimise cross population reporter ion interference. Graphical Abstract Highlights Revealed inflation of missing values as multiple TMT 10-plex batches are integrated. Analyzed the impact of integrating multiple TMT 10-plex batches on the quantification accuracy of both high and low abundance proteins. Established reliable detection of false positives caused by coisolation and reporter ion interference, highlighted by the incidence of Y chromosome peptides in all female channels. Optimized new experimental design set-ups to minimize cross population reporter ion interference via insights into coisolation and reporter ion interference. Multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent, tandem mass tags (TMT) in particular. Here we used a large iPSC proteomic experiment with twenty-four 10-plex TMT batches to evaluate the effect of integrating multiple TMT batches within a single analysis. We identified a significant inflation rate of protein missing values as multiple batches are integrated and show that this pattern is aggravated at the peptide level. We also show that without normalization strategies to address the batch effects, the high precision of quantitation within a single multiplexed TMT batch is not reproduced when data from multiple TMT batches are integrated. Further, the incidence of false positives was studied by using Y chromosome peptides as an internal control. The iPSC lines quantified in this data set were derived from both male and female donors, hence the peptides mapped to the Y chromosome should be absent from female lines. Nonetheless, these Y chromosome-specific peptides were consistently detected in the female channels of all TMT batches. We then used the same Y chromosome specific peptides to quantify the level of ion coisolation as well as the effect of primary and secondary reporter ion interference. These results were used to propose solutions to mitigate the limitations of multi-batch TMT analyses. We confirm that including a common reference line in every batch increases precision by facilitating normalization across the batches and we propose experimental designs that minimize the effect of cross population reporter ion interference.