Comparison of label-free quantification methods for the determination of protein complexes subunits stoichiometry

Protein complexes are the main molecular machines that support all major cellular pathways and their in-depth characterization are essential to understand their functions. Determining the stoichiometry of the different subunits of a protein complex still remains challenging. Recently, many label-free quantitative proteomic approaches have been developed to study the composition of protein complexes. It is therefore of great interest to evaluate these different methods in a stoichiometry oriented objective. Here we compare the ability of four absolute quantitative label-free methods currently used in proteomic studies to determine the stoichiometry of a well-characterized protein complex, the 26S proteasome

Deciphering preferential interactions within supramolecular protein complexes: the proteasome case

Intracellular protein breakdown is mainly performed by the Ubiquitin-Proteasome System in eukaryotic cells. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. They thus exhibit a very high heterogeneity whose function still needs to be understood. Here, using a new developed method based on the combination of affinity purification and protein correlation profiling associated with high resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub-complexes. In particular, we showed for the first time that the two main proteasome sub-types, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon-γ. The new method developed here constitutes an innovative and powerful strategy that could be widened to unravel the dynamic and heterogeneous nature of many other biologically relevant molecular systems

Label-Free Quantitative Proteomics Reveals the Dynamics of Proteasome Complexes Composition and Stoichiometry in a Wide Range of Human Cell Lines

The proteasome is the main proteolytic system involved in intracellular proteins homeostasis in eukaryotes. Although the structure of proteasome complexes has been well characterized, the distribution of its activators and associated proteins are less studied. Here, we determine the composition and the stoichiometry of proteasome complexes and their associated proteins in a wide range of human cell lines using a one-step affinity purification method and a label-free quantitative proteomic approach. We show that proteasome complexes are highly dynamic protein assemblies, the activity of which being regulated at different levels by variations in the stoichiometry of bound regulators, in the composition of catalytic subunits and associated proteins, and in the rate of the 20S catalytic core complex assembly

Correction to “Label-Free Quantitative Proteomics Reveals the Dynamics of Proteasome Complexes Composition and Stoichiometry in a Wide Range of Human Cell Lines”

Correction to “Label-Free Quantitative Proteomics Reveals the Dynamics of Proteasome Complexes Composition and Stoichiometry in a Wide Range of Human Cell Lines

Label-Free Quantitative Proteomics Reveals the Dynamics of Proteasome Complexes Composition and Stoichiometry in a Wide Range of Human Cell Lines

The proteasome is the main proteolytic system involved in intracellular proteins homeostasis in eukaryotes. Although the structure of proteasome complexes has been well characterized, the distribution of its activators and associated proteins are less studied. Here, we determine the composition and the stoichiometry of proteasome complexes and their associated proteins in a wide range of human cell lines using a one-step affinity purification method and a label-free quantitative proteomic approach. We show that proteasome complexes are highly dynamic protein assemblies, the activity of which being regulated at different levels by variations in the stoichiometry of bound regulators, in the composition of catalytic subunits and associated proteins, and in the rate of the 20S catalytic core complex assembly