Background: The architecture of eukaryotic cells is highly dynamic with protein complexes constantly being formed and resolved and proteins shuttling between different parts of the cell to carry out biological effects. Subcellular localization is one of the main regulatory levels of protein activity and protein interactions, however, a global view of cellular proteome organization remains relatively unexplored.
Purpose of the study: To develop a robust mass spectrometry based analysis pipeline to generate a proteome-wide view of subcellular localization.
Materials and Methods: We fractioned five different cell lines individually into five separate subcellular fractions and utilized high-resolution iso-electric focusing coupled to LC-MS/MS to generate in-depth proteomics data. Fractionation profiles were analyzed by tSNE and clustered by finite gaussian mixture models, finally, we used machine learning algorithms to classify proteins into subcellular compartments.
Results: Our analysis gives a highly accurate classification of proteins mapping to 12,418 individual genes into cytosolic, nuclear, secretory and mitochondrial locations and further into 15 specific subcellular compartments. We also investigated the impact of alternative splicing and protein domains on localization, complex member co-localization as well as protein relocalization after growth factor inhibition. Additionally, we have combined the method with peptide level phospho-enrichment which allows for identification of PTM-dependent localization.
Conclusion: Our analysis provides knowledge about the cellular architecture and the complexity of the spatial organization of the proteome, as we show that the majority of proteins have a single main subcellular localization, that alternative splicing rarely affects subcellular localization and that cell types are best distinguished by expression of proteins exposed to the surrounding environment.