Multiplexed complexome profiling using tandem mass tags

Abstract

Complexome profiling is a rapidly spreading, powerful technique to gain insight into the nature of protein complexes. It identifies and quantifies protein complexes separated into multiple fractions of increasing molecular mass using mass spectrometry-based, label-free bottom-up proteomics. Complexome profiling enables a sophisticated and thorough characterization of the composition, molecular mass, assembly, and interactions of protein complexes. However, in practice, its application is limited by the large number of samples it generates and the related time of mass spectrometry analyses. Here, we report an improved process workflow that implements tandem mass tags for multiplexing complexome profiling. This workflow substantially reduces the number of samples and measuring time without compromising protein identification or quantification reliability. In profiles from mitochondrial fractions of cells recovering from chloramphenicol treatment, tandem mass tags-multiplexed complexome profiling exhibited migration patterns of mature ATP synthase (complex V) and assembly intermediates that were consistent in composition and abundance with profiles obtained by the label-free approach. Reporter ion quantifications of proteins and complexes unaffected by the chloramphenicol treatment presented less variation in comparison to the label-free method. Incorporation of tandem mass tags enabled an efficient and robust complexome profiling analysis and may foster broader application for protein complex profiling in biomedical research and diagnostics.