Combining LOPIT with differential ultracentrifugation for high-resolution spatial proteomics

Abstract

The study of protein localisation has greatly benefited from high-throughput methods utilising cellular fractionation and proteomic profiling. Hyperplexed Localisation of Organelle Proteins by Isotope Tagging (hyperLOPIT) is a well-established method in this area. It achieves high-resolution separation of organelles and subcellular compartments but is relatively time- and resource-intensive. As a simpler alternative, we here develop Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) and compare this method to the density gradient-based hyperLOPIT approach. We confirm that high-resolution maps can be obtained using differential centrifugation down to the suborganellar and protein complex level. HyperLOPIT and LOPIT-DC yield highly similar results, facilitating the identification of isoform-specific localisations and high-confidence localisation assignment for proteins in suborganellar structures, protein complexes and signalling pathways. By combining both approaches, we present a comprehensive high-resolution dataset of human protein localisations and deliver a flexible set of protocols for subcellular proteomics.

Integrated multi-omics reveals common properties underlying stress granule and P-body formation

“…This supports the definition of an elution profile across multiple fractions ( Fig. 2) that we argue better represents the true subcellular body; much like the LOPIT approach on which it is based (Geladaki, Kocevar Britovsek et al, 2019). In turn, the downstream fuzzy clustering has supported the identification of a previously unrecognized segregation pattern for associated RNA binding proteins.…”

“…An approach was adapted from the hyperLOPIT protocol developed by Lilley and colleagues (Geladaki et al, 2019) for identification of members of HMW complexes, substituting fractions collected from the centrifugation and immunoprecipitation steps for ultracentrifugation. First, the MaxQuant 'peptides.txt' output file was processed using the MSnbase R packge (Gatto & Lilley, 2012).…”

A subcellular atlas ofToxoplasmareveals the functional context of the proteome

“…Fifty 1-min fractions were collected along the elution profile of the peptides (approximately from minute 10 to 60 of the program) and reduced to dryness. For the downstream LC-MS analysis, the fractions corresponding to each TMT10plex set were concatenated into 15-18 samples by combining pairs of fractions which eluted at different time points during the gradient, e.g., fraction 1, 16, and 31, fraction 2, 17, and 32, etc. LC-MS analysis of peptides All mass spectrometry analyses were performed on an Orbitrap Fusion™ Lumos™ Tribrid™ instrument coupled to a Dionex Ultimate™ 3000 RSLCnano system (Thermo Fisher Scientific) as described in (Geladaki et al, 2019).…”

Thermal proteome profiling for interrogating protein interactions

“…The advent of mass spectrometry-based proteomics has transformed the study of protein biology, by allowing for a global view of the proteome in its native context (Aebersold & Mann, 2016). This encompasses, for example, the study of protein abundances (Kim et al, 2014;Wilhelm et al, 2014), turnover (Schwanhausser et al, 2011), localization (Geladaki et al, 2019), or post-translational modifications (Potel et al, 2018). Recently, biophysical properties of proteins have been explored and studied system-wide with proteomics approaches.…”