Application of quantitative cross-linking mass spectrometry methods to study interactome differences.

Abstract

Proteins carry out the vast majority of biological function inside living cells. They do so by changing their shapes, interacting with one another and other molecules such as DNA and RNA. Gaining an understanding of intra and inter molecular protein interactions on a systems-level would allow for much deeper understanding of how biological functions are performed and regulated. Quantitative comparisons of these interactions between different systems or upon perturbations can increase understanding of remodeling associated with the aging, disease, and treatments. Here, cross-linking mass spectrometry is demonstrated with quantitative comparisons of protein interactions within intact systems, such as cells and membrane bound organelles. This work shows that reproducible remodeling of interactomes associated with aging in skeletal muscle mitochondria can be detected with isobaric quantitative protein interaction reporter (iqPIR) technologies. Moreover, changes in the interactome showed correlation with age-associated mitochondrial functional decline. Interactome differences associated with distinct functional differences in different cell types were also observed. Quantitative comparison of interactomes of human cell lines HEK293, HeLa and MCF7 uncovered differences in chromatin remodeling, mitochondrial transport and others that are independent of protein abundance levels. Utilizing a novel genetic mouse model that allows isolation of mitochondria from tubule or podocyte kidney cells, quantitative cross-linking and mass spectrometry enabled identification of differentially regulated proteins and pathways in mitochondria within these cell types that would otherwise remain unknown.