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Localization of Organelle Proteins by Isotope Tagging (LOPIT): past, present and future

28 May 2020 4:42 PM | Anonymous

Mohamed Elzerk and Kathryn S. Lilley, Cambridge Centre for Proteomics, Department of Biochemistry, The Milner Therapeutics Institute, University of Cambridge, Cambridge, UK

The interior of eukaryotic cells is characterised by a high degree of structural and functional partitioning into distinct microenvironments dedicated to diverse and specific roles. Trafficking between subcellular niches allows proteins to drive biological processes such as maintaining homeostasis and regulating stress response. Aberrant trafficking is known to be the root of many diseases 1,2. Methods such as microscopy or affinity tagging are essential to determine the location of individual proteins or protein repertoires of purified organelles 3,4. However, a thorough understanding of functional dynamics of the proteome, requires a high throughput ability to define the spatial context of the entire proteome of the cell across different cell types, conditions and time points. Many of the current spatial proteomics techniques have been inspired by the protein correlation profiling principle exploited by cell biologists in the 1950 and 1960 to uncover new organelle 5–12. Membrane bound organelles and protein complexes co-fractionate upon centrifugation purely on the basis of their physical properties such as size, shape and density. Proteins with similar distributions to those exhibited by organelle marker proteins are assigned a single or multiple locations. Since its inception, the Cambridge Centre for Proteomics has contributed extensively to the establishment of spatial proteomics as a field primarily through the development of a technique known as Localization of Organelle Proteins by Isotope Tagging (LOPIT).

The LOPIT approach combines organelle separation based on their characteristic buoyant densities or sedimentation rate by ultracentrifugation, with quantitative proteomics employing multiplexed by in vitro stable isotope labelling, highly sensitive mass spectrometers and multivariate statistical analysis (figure 1). Dunkley et al published the first draft of LOPIT In 2004, providing localisation annotations in Arabidopsis thaliana using Isotope-coded affinity tag (ICAT)5. A partial least squares-discriminant analysis (PLS-DA) algorithm enabled novel localisation of a number of proteins to ER, Golgi, and mitochondrial/plastid. Subsequently, the LOPIT methodology has evolved with the development of the field of mass spectrometry-based proteomics with the emergence of the multiplexing capacity of isotope labelling and the higher resolution of Orbitrap mass spectrometers. Furthermore, data analysis and visualisation tools have been tailored towards the output of LOPIT analysis. The pRoloc and pRolocGUI R packages cover a broad range of computational methods from unsupervised, supervised and semi-supervised machine learning, novelty detection and cluster separation assessment to, more recently, transfer learning and Bayesian modelling 13,14. Over the years, the applications of LOPIT extended to multiple biological systems, most recently the first protein atlas of Toxoplasma gondii 15–19.

In 2016, Christoforou and colleagues exquisitely portrayed the protein map of mouse stem cells in a single experiment15. This improved version of LOPIT was rebranded as hyperplexed LOPIT (hyperLOPIT) thanks to higher multiplexing capabilities of amine-reactive tandem mass tags (TMT) 10-plex, MS/MS acquisition using synchronous precursor selection to improve quantitative accuracy and application of support vector machines (SVM) for data analysis. Almost half of the mouse stem cell proteome were annotated to multiple subcellular locations which was later also supported by the human cell atlas project3. Moreover, hyperLOPIT enabled subcellular localisation of some protein isoforms, protein complexes and signalling pathways. A year ago, a comparable system-wide resolution was also obtained differential ultracentrifugation based LOPIT, or LOPIT-DC, in which the spatial proteome of human osteosarcoma U-2 OS cell line was fully characterised using less time, material and resources18.

Figure 1: A schematic overview of HyperLOPIT (left) and LOPIT-DC (right) workflows

Membrane trafficking is an exemplar field which could benefit greatly from LOPIT. In particular, retrograde trafficking from endosomes to the trans-Golgi network (TGN) involves multiple partially redundant pathways that generate distinct pools of vesicles which are difficult to purify using other antibodies based techniques. Recently, Shin et al applied LOPIT-DC to characterise the endosome-to-Golgi vesicles that are selectively captured by golgin tethers at the TGN19. These golgins were ectopically redirected to mitochondria in order to determine the content of the specific endosome-to-Golgi vesicles they capture. Bayesian non-parametric testing was employed to identify protein movements towards the mitochondria. A profile shift of 45 transmembrane proteins and 51 peripheral membrane proteins of the endosomal network were detected including known cargo and trafficking machinery of the clathrin/AP-1, retromer-dependent and -independent transport pathways.These findings opens the exciting prospect of using LOPIT to interrogate dynamic spatial protein movements.

Spatial proteomics is still an emerging technology. The continual development of multiplexed mass spectrometry analysis promises enhanced subcellular resolution and motivating dynamic protein localisation studies while alleviating the technical variability 20. Furthermore, LOPIT is a modular technique which facilitates the use of complementary techniques such as RNA sequencing and metabolic profiling. The combination of LOPIT with transcriptomic and metabolic profiling in a spatial multi-omics map has the capacity to drastically reshape our understanding of cell biology. In conclusion, LOPIT has been substantially developed to be a user-friendly approach with the availability of detailed online experimental protocols and an open-source bioinformatics suite 14,21,22. We encourage our readers to consider applying workflows such as LOPIT to their experiments to harness the power of spatial proteomics.


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