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Rapid Profiling of Protein Complex Reorganization in Perturbed Systems.

ABSTRACT: Protein complexes constitute the primary functional modules of cellular activity. To respond to perturbations, complexes undergo changes in their abundance, subunit composition, or state of modification. Understanding the function of biological systems requires global strategies to capture this contextual state information. Methods based on cofractionation paired with mass spectrometry have demonstrated the capability for deep biological insight, but the scope of studies using this approach has been limited by the large measurement time per biological sample and challenges with data analysis. There has been little uptake of this strategy into the broader life science community despite its rich biological information content. We present a rapid integrated experimental and computational workflow to assess the reorganization of protein complexes across multiple cellular states. The workflow combines short gradient chromatography and DIA/SWATH mass spectrometry with a data analysis toolset to quantify changes in a complex organization. We applied the workflow to study the global protein complex rearrangements of THP-1 cells undergoing monocyte to macrophage differentiation and subsequent stimulation of macrophage cells with lipopolysaccharide. We observed substantial proteome reorganization on differentiation and less pronounced changes in macrophage stimulation. We establish our integrated differential pipeline for rapid and state-specific profiling of protein complex organization.

SUBMITTER: Bludau I

PROVIDER: S-EPMC10167687 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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Rapid Profiling of Protein Complex Reorganization in Perturbed Systems.

Bludau Isabell I Nicod Charlotte C Martelli Claudia C Xue Peng P Heusel Moritz M Fossati Andrea A Uliana Federico F Frommelt Fabian F Aebersold Ruedi R Collins Ben C BC

Journal of proteome research 20230414 5

Protein complexes constitute the primary functional modules of cellular activity. To respond to perturbations, complexes undergo changes in their abundance, subunit composition, or state of modification. Understanding the function of biological systems requires global strategies to capture this contextual state information. Methods based on cofractionation paired with mass spectrometry have demonstrated the capability for deep biological insight, but the scope of studies using this approach has ...[more]

PMID: 37058003

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Project description:Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.

Project description:BackgroundInvasive aedine mosquito species have become a major issue in many parts of the world as most of them are recognised vectors or potentially involved in transmission of pathogens. Surveillance of these mosquitoes (e.g. Ae. aegypti, Yellow fever mosquito, Aedes albopictus, Asian tiger mosquito) is mainly done by collecting eggs using ovitraps and by identification of the larvae hatched in the laboratory. In order to replace this challenging and laborious procedure, we have evaluated matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) for easy and rapid species identification.MethodsIndividual protein profiles were generated using five eggs each of nine aedine species (Ae. aegypti, Ae. albopictus, Ae. atropalpus, Ae. cretinus, Ae. geniculatus, Ae. japonicus, Ae. koreicus, Ae. phoeniciae, Ae. triseriatus) from various geographical origins, and species-specific biomarker mass sets could be generated. A blinded validation using our reference data base for automated egg identification was performed. In addition, pools of 10 aedine eggs (132 two-species and 18 three-species pools) in different ratios were evaluated.ResultsSpecific biomarker mass sets comprising 18 marker masses could be generated for eggs of nine container-inhabiting aedine species, including all the major invasive and indigenous species of Europe and North America. Two additional masses shared by all investigated aedine species are used as internal calibrators. Identification of single eggs was highly accurate (100% specificity, 98.75% sensitivity), and this method is also of value for the identification of species in pools of ten eggs. When mixing two or three species, all were identified in all pools in at least 2 or 1 of the 4 loaded replicates, respectively, if the "lesser abundant" species in the pool accounted for three or more eggs.ConclusionsMALDI-TOF MS, which is widely applied for routine identification of microorganisms in clinical microbiology laboratories, is also suited for robust, low-cost and high throughput identification of mosquito vectors in surveillance programmes. This tool can further be developed to include a wide spectrum of arthropods but also other Metazoa for which surveillance is required, and might become the method of choice for their centralised identification via online platforms.

Dataset Information

Rapid Profiling of Protein Complex Reorganization in Perturbed Systems.

Publications

Rapid Profiling of Protein Complex Reorganization in Perturbed Systems.

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