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 post-translational 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.
Highlights
Knowledge of the subcellular distribution of proteins is vital for understanding cellular mechanisms
Our understanding of the biology of embryonic stem (ES) cells is deeply rooted in our knowledge of their transcriptomes, epigenetics and underlying gene regulatory networks, which have created a foundation for understanding pluripotency and the transition to differentiation[3,4]
The resulting peptides are differentially labelled with amine-reactive tandem mass tag (TMT) reagents[13], which allow peptides derived from each fraction to be distinguished by mass spectrometry
Summary
Knowledge of the subcellular distribution of proteins is vital for understanding cellular mechanisms. Immunocytochemistry is dependent on the availability of high-specificity and high-sensitivity antibodies, while fluorescent fusion proteins are vulnerable to aberrant localization due to the effect of the fusion moiety on protein topology[9,10]. These limitations can be overcome with complementary technologies such as protein mass spectrometry (MS), which offers the capability to assay thousands of proteins simultaneously and in their native state[11]. Localization of organelle proteins by isotope tagging (LOPIT) is a quantitative proteomics method for the high throughput and simultaneous characterization of multiple subcellular compartments, without the requirement for total purification of compartments of interest[12]. Classification algorithms are used to assign proteins to subcellular compartments based on correlation with organellar marker proteins
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