Hi all!

I've run both LFQ and TMT 18-plex proteomics on the same protein extracts.

My experiment consists of two study conditions, and 8 biological replicates.

After digesting my protein extractions, I ran half of the peptide preparation using DDA with four technical replicates, and the other half I TMT tagged (18-plex, two reference channels, one mixture) fractionated, and ran using an SPS MS3 method on the Fusion Lumos.

I've done the searches in PD2.4, and summarised the results with `MSstats` and `MSstatsTMT`.

I'm currently working on how to deal with two different datasets of the same experiment, the original plan was to use the LFQ dataset for the improved coverage, and the TMT dataset for improved quantification.

One thing I've noticed is that while the TMT dataset has significantly better adjusted p-values, the fold changes are less pronounced than the LFQ dataset, meaning that quite a few proteins fail the biological significance thresholds. See the attached volcano plots (vertical dotted lines represent 0.58 log2 FC, horizontal 0.05 adjusted p-value). The scales are not consistent between the plots sorry!

I'm aware that MS2 TMT methods have an issue with reporter ion compression blunting fold change values, and was hoping that it would be less of an issue with my MS3 method. Is there a correction for this, or does this reflect a lack of dramatic fold-change in my biology?

Any other tips for integrating LFQ and TMT data would also be appreciated!

Sam