Biology subjects

Foo, B.

Publications and source records attributed to Foo, B..

2 recordsLinked to original sources

bioRxiv2024

mini-Complexome Profiling (mCP), an FDR-controlled workflow for global targeted detection of protein complexes

IntroductionCo-fractionation mass spectrometry couples native-like separations of protein/protein complexes with mass spectrometric proteome analysis for global characterization of protein networks. The technique allows for both de novo detection of complexes and for the detection of subtle changes in their protein composition. The typical requirement for fine-grained fractionation of >80 fractions, however, translates into significant demands on sample quantity and mass spectrometric instrument time, and represents a significant barrier to experimental replication and the use of scarce sample material (ex. Patient biopsies). MethodsWe developed mini-Complexome Profiling (mCP), a streamlined workflow with reduced requirements for fractionation and, thus, biological material and laboratory and instrument time. Soluble and membrane-associated protein complexes are extracted from biological material under mild conditions, and fractionated by Blue Native electrophoresis using commercial equipment. Each fraction is analyzed by data independent acquisition mass-spectrometry, and known protein complexes are detected based on the coelution of known components using a novel R package with a controlled false discovery rate approach. The tool is available to the community on a GitHub repository. ResultsmCP was benchmarked using HEK293 cell lysate and exhibited performance similar to established workflows, but from a significantly reduced number of fractions. We then challenged mCP by performing comparative complexome analysis of cardiomyocytes isolated from different chambers from a single mouse heart, where we identified subtle chamber-specific changes in mitochondrial OxPhos complexes. DiscussionThe reduced sample and instrument time requirements open up new applications of co-fractionation mass spectrometry, specifically for the analysis of sparse samples such as human patient biopsies. The ability to identify subtle changes between similar tissue types (left/right ventricular and atrial cardiomyocytes) serves as a proof of principle for comparative analysis of mild/asymptomatic disease states.

systems biology↗

bioRxiv2024

Unbiased complexome profiling and global proteomics analysis reveals mitochondrial impairment and potential changes at the intercalated disk in presymptomatic R14Delta/+ mice hearts

BackgroundPhospholamban (PLN) is a sarco-endoplasmic reticulum (SER) membrane protein that regulates cardiac contraction/relaxation by reversibly inhibiting the SERCA2a Ca2+-reuptake pump. The R14{Delta}-PLN mutation causes severe cardiomyopathy that is resistant to conventional treatment. Protein complexes and higher-order supercomplexes such as intercalated disk components and Ca+2-cycling domains underlie many critical cardiac functions, a subset of which may be disrupted by R14{Delta}-PLN. MethodsWe developed an improved complexome profiling (CP) workflow specifically geared towards identifying disruption of very high molecular-weight (>2 MDa) protein complexes and supercomplexes in presymptomatic R14{Delta}/+ mice hearts. Ventricular tissues were homogenized under non-denaturing conditions, fractionated by size-exclusion chromatography (SEC) and subjected to quantitative data-independent acquisition mass spectrometry (DIA-MS) proteomics analysis. Systematic analysis of CP data using conventional strategies yielded limited insights, likely due to underrepresentation of cardiac-specific complexes in the curated protein complex databases used as ground-truth for analysis. We thus developed PERCOM: a novel data analysis strategy that does not rely upon protein complex databases and can, furthermore, be implemented on widely available spreadsheet software. ResultsSEC-DIA-MS coupled with PERCOM identified 296 proteins with disrupted elution profiles in presymptomatic 28wk-old R14{Delta}/+ mice. Hits were significantly enriched for mitochondrial and intercalated disk (ICD) components. Alterations to mitochondrial and ICD supercomplexes were observed in mice as young as 9wks of age and were associated with reduced expression of mitochondrial proteins and maximal oxygen consumption rate. ConclusionUsing a novel CP workflow, we identify mitochondrial alterations as an early-stage R14{Delta}-PLN event and provide preliminary data showing effects at the ICD. These molecular components underlie critical cardiac functions and their alteration at a young age may contribute to R14{Delta}-PLN pathogenesis.

cell biology↗