Abstract:
Knowledge of the subcellular distribution of proteins is vital for understanding cellular
mechanisms. Capturing the subcellular proteome in a single experiment has proven
challenging, with studies focusing on specific compartments or assigning proteins to
subcellular niches with low resolution and/or accuracy. Here we introduce hyperLOPIT, a
method that couples extensive fractionation, quantitative high-resolution accurate mass
spectrometry with multivariate data analysis. We apply hyperLOPIT to a pluripotent stem cell
population whose subcellular proteome has not been extensively studied. We provide
localization data on over 5,000 proteins with unprecedented spatial resolution to reveal the
organization of organelles, sub-organellar compartments, protein complexes, functional
networks and steady-state dynamics of proteins and unexpected subcellular locations. The
method paves the way for characterizing the impact of post-transcriptional and posttranslational
modification on protein location and studies involving proteome-level locational
changes on cellular perturbation. An interactive open-source resource is presented that
enables exploration of these data.
