Quantitative density gradient analysis by mass spectrometry (qDGMS) and complexome profiling analysis (ComPrAn) R package for the study of macromolecular complexes

Abstract

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.

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

“…A recent critical appraisal exposed a surprisingly high false discovery rate of global complexomic analyses, when complex membership is called by direct matching of protein distributions (Shatsky et al, 2016). This happens despite the ever more sophisticated bioinformatic pipelines used for the analysis of complexomic data (Dong et al, 2008;Havugimana et al, 2012;Kristensen et al, 2012;Giese et al, 2014;Gazestani et al, 2016;Páleníková et al, 2021a). The problem comes, on the one side, from natural spurious overlapping of unrelated profiles, and on the other, from the general analytical caveat of matching algorithms: most statistical measures are inherently tailored to look for differences, not similarities (Motulsky, 2010).…”

“…Other separation techniques, such as clear native PAGE, sucrose gradient centrifugation, and size exclusion chromatography, have been successfully used to partition complexes by size and shape (Andersen et al, 2003;Peltier et al, 2006;Hartman et al, 2007;Chen and Williamson, 2013;Kirkwood et al, 2013;Larance et al, 2016;Páleníková et al, 2021a). However, the majority of complexomic studies now rely on a combination of orthogonal fractionation methods, thus decreasing the probability of chance co-elution for proteins involved in closely migrating but otherwise unrelated assemblies.…”

“…Though largely focused on proteins, some complexomic studies had already reserved a place of honor to major RNPs, most often ribosomes (Wessels et al, 2013;Gazestani et al, 2016;Larance et al, 2016;Chatzispyrou et al, 2017;Van Strien et al, 2019;Páleníková et al, 2021a). These works resulted in several interesting findings, including details of bacterial and mitochondrial ribosome assembly (Chen and Williamson, 2013;Sashital et al, 2014;Davis et al, 2016;Rugen et al, 2019) and some of the first observations of CRISPR ribonucleoproteins FIGURE 2 | Major complexomic approaches.…”

A late-stage assembly checkpoint of the human mitochondrial ribosome large subunit

“…To investigate the mechanistic basis for the defect in mitochondrial translation, the composition of mitochondrial ribosomes was assessed using quantitative density gradient analysis by mass spectrometry -qDGMS (Páleníková et al, 2021). While no shifts in the sedimentation profile of mitoribosomal components were observed, proteins of the mtLSU were enriched upon MRM2 depletion (Figure 4 and Figure S4), indicating the accumulation of mature or near-mature forms of this subunit.…”

“…The procedure followed that described for quantitative analysis of density gradients by mass spectrometry (qDGMS) in (Páleníková et al, 2021). In brief, cells were cultured in DMEM for SILAC, supplemented with 20% dialysed FBS, 50 mg mL -1 uridine, 1x penicillin-streptomycin, 200 mg L -1 L-proline, and either 0.398 mM L-arginine and 0.798 mM L-lysine, or 0.398 mM 13 C6, 15 N4-L-arginine and 0.798 mM 13 C6, 15 N2-Llysine.…”