Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS
Volume 1862, Issue 6, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.bbabio.2021.148399
Keywords
Proteomics; Density gradient ultracentrifugation; Complexome profiling; Mitochondrial ribosome; SILAC; R package
Categories
Funding
- Medical Research Council, UK [MC_UU_00015/4]
- Marie Sklodowska-Curie ITN-REMIX grant [721757]
- Fundacao para a Ciencia e a Tecnologia, Portugal [PD/BD/105750/2014]
- Marie Curie Actions (MSCA) [721757] Funding Source: Marie Curie Actions (MSCA)
- MRC [MC_UU_00015/4] Funding Source: UKRI
- Fundação para a Ciência e a Tecnologia [PD/BD/105750/2014] Funding Source: FCT
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The method described utilizes density separation and mass spectrometric quantitation for quantitative analysis of native protein or ribonucleoprotein complexes, with the ComPrAn software package providing tools for data organization, analysis, and visualization. This technology can be applied to study the assembly and dynamic properties of macromolecular complexes.
Many cellular processes involve the participation of large macromolecular assemblies. Understanding their function requires methods allowing to study their dynamic and mechanistic properties. Here we present a method for quantitative analysis of native protein or ribonucleoprotein complexes by mass spectrometry following their separation by density - qDGMS. Mass spectrometric quantitation is enabled through stable isotope labelling with amino acids in cell culture (SILAC). We provide a complete guide, from experimental design to preparation of publication-ready figures, using a purposely-developed R package - ComPrAn. As specific examples, we present the use of sucrose density gradients to inspect the assembly and dynamics of the human mitochondrial ribosome (mitoribosome), its interacting proteins, the small subunit of the cytoplasmic ribosome, cytoplasmic aminoacyl-tRNA synthetase complex and the mitochondrial PDH complex. ComPrAn provides tools for analysis of peptide-level data as well as normalization and clustering tools for protein-level data, dedicated visualization functions and graphical user interface. Although, it has been developed for the analysis of qDGMS samples, it can also be used for other proteomics experiments that involve 2-state labelled samples separated into fractions. We show that qDGMS and ComPrAn can be used to study macromolecular complexes in their native state, accounting for the dynamics inherent to biological systems and benefiting from its proteome-wide quantitative and qualitative capability.
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