Spatial proteomics: mapping cell signalling pathways at sub-cellular resolution

A new collaborative study involving NAPI partner Fridtjof Lund-Johansen describes a novel approach to perform mass spectrometry-based analysis of proteins across different cell compartments.

Schematic for the spatial proteomics workflow developed in collaboration with researchers at the University of Copenhagen

Each of our cells possesses a complex machinery of protein molecules that act in concert to regulate cell behaviour. Responding to a range of stimuli, protein molecules interact with one another to regulate each other’s conformation, modification status, activity and sub-cellular localisation. A sequence of such interactions – a ‘signalling pathway’ – can culminate in an important response by the cell. For example, when a growth factor binds to receptors on a cell’s surface, signalling pathways are triggered that traverse various cell compartments, ultimately reaching the nucleus where the transcription of genes that promote cell division is activated.

Tracking protein localisation by mass spectrometry

Scientists have studied cell signalling pathways for many decades, including how deregulation of certain pathways can lead to human diseases such as cancer. Mass spectrometry (MS)-based proteomics provides a powerful tool for these studies, as it enables systems-level analyses of many thousands of proteins in one go. MS can also be used to assess differences in protein levels between samples, as well as detect protein modifications, such as phosphorylation, that play important roles in cell signalling.

Corresponding authors Fridtjof Lund-Johansen (centre) and Jesper Olsen (right), and first author Ana Martinez-Val (left)

However, to map signalling pathways comprehensively, it is important to be able to track protein localisation over time. This provides a visual of protein movement from one cell compartment to another (leading to interaction with, and regulation of, new partners, thereby relaying the signal). Towards this goal, researchers have used sub-cellular fractionation techniques, which separate out cellular compartments based on different properties such as density or solubility. Subsequent MS analysis of individual sub-cellular compartments – referred to as ‘spatial proteomics’ – provides the extra dimension of protein localisation when compared to a single analysis of whole cells, and therefore a higher resolution image of cell signalling pathways is achieved.

Improving spatial proteomics capabilities

In a recent collaborative study, NAPI partner Fridtjof Lund-Johansen teamed up with researchers from the University of Copenhagen, Denmark, to develop a new approach for spatial proteomics. This approach enables both a) deep coverage of cellular proteins, and b) relatively short and simple sample processing and MS analysis.

The researchers believe this marks an important improvement over previous spatial proteomics strategies, which either achieve deep protein coverage at the expense of laborious and time-consuming methodologies, or provide quicker and more practical approaches at the expense of detecting fewer proteins from cells. The new approach thus provides the ‘best of both worlds’, such that researchers can assess sub-cellular protein distribution with not just high resolution but also high throughput, meaning multiple samples can be analysed in a single experiment. This allows the inclusion of more experimental conditions (e.g. multiple time points to assess temporal changes in sub-cellular protein localisation), as well as more biological repeat analyses (improving confidence in the results).

Splitting the cell in six

Dr Lund-Johansen was involved in developing the new sub-cellular fractionation strategy. Explaining the approach, he said:

“As an initial test, we used HeLa cervical cancer cells to see how detailed a picture of protein signalling we could achieve, and we performed four biological repeat experiments to assess the reproducibility of our approach. We treated the cells sequentially with six extraction buffers that differed in their salt and chemical composition (see schematic at the top of this page). This resulted in six fractions corresponding to different cellular compartments – for example proteins associated with the cytosol and cytoskeleton were in the first two fractions, whilst nuclear proteins were in the last two fractions. Each fraction was then analysed by our colleagues in Copenhagen using highly sensitive MS. Importantly, in addition to assessing protein content, their approach also enabled the detection of phosphorylation sites in each fraction, adding an extra layer of information about the signalling pathways functioning within different parts of the cell”.

Sensitive sub-cellular detection of protein and phosphorylation sites

Proteins (left) and phosphosites (right) identified from HeLa samples. Bar height represents mean number of identifications form 4 experimental replicates. Error bars represent standard deviation in identification numbers between replicates.

This preliminary approach led to the detection of approximately 7000 proteins and 8000 phosphorylation sites from the HeLa cells (see image right) – an impressive depth of coverage considering the entire MS analysis time was less than one day (approx 20 hours for all four biological repeats, whilst the same analysis would take up to a week of MS time with other published approaches). The approach also demonstrated good levels of reproducibility, with more than 90% of proteins found in the same sub-cellular fraction in at least three out of four replicates.

Interestingly, while the number of proteins was highly similar in each fraction (average 4000), the distribution of phosphorylation events was more variable, with fractions three (containing membrane proteins) and six (containing nucleolar proteins) exhibiting substantially lower levels of phosphorylation sites than the other four fractions. This may, in part, be explained by lower levels of the kinases that carry our phosphorylation in these fractions. Indeed, fewer kinases were detected in the membrane compartment of HeLa cells in this analysis.

Answering important biological questions

To demonstrate the power of their methodology to provide novel insights into important biological questions, the researchers next performed larger-scale experiments to measure cellular signalling events activated downstream of different stimuli.

Firstly, they investigated the response to epidermal growth factor (EGF) stimulation, which is known to promote cell growth and replication and therefore play an important role in many biological processes, as well as disease mechanisms.

The researchers performed a time course experiment, analysing six fractions from cells exposed to EGF for 0, 2, 8, 20 and 90 minutes (four biological repeats for each time point; see image below). Collectively, this facilitated a dynamic and comprehensive depiction the cellular response to EGF, including how protein and phosphorylation site levels changed across time and sub-cellular space. In particular, the short time points enabled insights into the rapid signalling events that immediately follow EGF exposure. For example, important signalling proteins were recruited from the cytosol to the cell membrane-bound EGF receptor within two minutes, phosphorylated as part of their role in the signalling pathway, and then shuttled back to the cytosol after 20 minutes. This level of detail would not be possible by looking at limited numbers of time points after EGF stimulation, again highlighting the benefit of the high-throughput approach that allows multiple samples to be analysed.

Left: schematic for the EGF time course spatial proteomics workflow. Right: A selection of phosphorylation sites in EGF-induced signalling pathways. The novel workflow allows tracking of phosphorylation events across time and space (e.g. EGFR phosphotyrosine events occur only at the cell membrane and peak between 0-20 mins).

Importantly, the new spatial proteomics method is not limited to analysing cells in culture. The researchers also performed EGF analyses in vivo on samples taken from mice. Analysing the livers and kidneys of mice injected with EGF or, as a control, saline, the researchers found that most of the results observed for HeLa cells were reproduced in living tissues, although interesting differences in mitochondria – the energy-generating ‘bateries’ of our cells – were observed between liver and kidney cells.

In addition to studying EGF-induced signalling, the researchers also performed spatial proteomics analysis of cells exposed to hyperosmotic stress, which involves the rapid movement of water across a cell’s membrane and is known to cause protein translocation between cell compartments. This revealed that the large subunit of ribosomes (where mRNA is translated into proteins) are transported from cytosolic to nuclear compartments by osmotic stress. Further analysis revealed this was due to an alteration in the production and assembly of ribosome subunits, such that they become trapped and accumulate in the nucleus. These findings help develop a more comprehensive overview of the mechanisms regulating response to cellular stress, which is thought to contribute to many human diseases, such as cancer or inflamatory conditions.

Useful tool, useful data

The spatial proteomics approach can be applied to in vivo studies, as demonstrated by the analysis of livers and kideneys isolated from mice treated with EGF or, as a control, saline.

Overall, the new spatial proteomics technique represents a highly valuable tool for those looking to generate comprehensive systems-wide maps of the protein and phosphoprotein dynamics that control cell behaviour. Furthermore, the large datasets generated in this publication will provide a valuable resource for those researchers interested in sub-cellular distribution of proteins. This is something Dr Lund-Johansen and his colleagues have already considered:

“Our cell culture and mouse model datasets have been made available in an easily-accessible and navigable web-database form. This has a simple user interface that allows people to search for their proteins of interest and investigate which cellular compartments they belong to, as well as whether their phosphorylated forms are found in those same compartments. We hope this will be a valuable tool for many future studies investigating a range of biological topics”.

The web-database connected to this publication is freely accessible on the spatio-temporal dymanics website. You can also find a user manual and excel file for MetaMass – a tool for meta-analysis of sub-cellular proteomics data used in the publication – on the resources page of the NAPI website.

Fridtjof Lund-Johansen and lead author Ana Martinez-Val both presented analysis tools used in this study as part of the first NAPI bioinformatics workshop earlier this year. To be kept informed of future workshops please contact the NAPI administrative manager.

You can read the paper in full on the Nature Communications website.


Published Dec. 14, 2021 10:44 AM - Last modified Dec. 14, 2021 10:44 AM