MSstatsTMT: Statistical detection of differentially abundant proteins in experiments with isobaric labeling and multiple mixtures.

Huang, Ting; Choi, Meena; Tzouros, Manuel; Golling, Sabrina; Pandya, Nikhil Janak; Banfai, Balazs; Dunkley, Tom; Vitek, Olga

Tandem mass tag (TMT) is a multiplexing technology widely-used in proteomic research. It enables relative quantification of proteins from multiple biological samples in a single mass spectrometry run with high efficiency and high throughput. However, experiments often require more biological replicates or conditions than can be accommodated by a single run, and involve multiple TMT mixtures and multiple runs. Such larger-scale experiments combine sources of biological and technical variation in patterns that are complex, unique to TMT-based workflows, and challenging for the downstream statistical analysis. These patterns cannot be adequately characterized by statistical methods designed for other technologies, such as label-free proteomics or transcriptomics. This manuscript proposes a general statistical approach for relative protein quantification in mass spectrometry-based experiments with TMT labeling. It is applicable to experiments with multiple conditions, multiple biological replicate runs and multiple technical replicate runs, and unbalanced designs. It is based on a flexible family of linear mixed-effects models that handle complex patterns of technical artifacts and missing values. The approach is implemented in MSstatsTMT, a freely available open-source R/Bioconductor package compatible with data processing tools such as Proteome Discoverer, MaxQuant, OpenMS and SpectroMine. Evaluation on a controlled mixture, simulated datasets, and three biological investigations with diverse designs demonstrated that MSstatsTMT balanced the sensitivity and the specificity of detecting differentially abundant proteins, in particular in large-scale experiments with multiple biological mixtures.