Are you familiar with proteomics, the science that focuses on the proteome, the entire set of proteins in the human body? In Rennes, the Protim platform team is actively working to advance knowledge in this field.
"Twenty years ago, when the major human genome sequencing program was announced, we thought that once it was completed, we would know everything about human diseases. This was really a figure of speech because actually it is not the gene that carries the function, but the protein!" explains Charles Pineau, who heads up the Protim platform*. For example, we have around the same number of genes as a mouse or a Caenorhabditis elegans worm, but the proteins they encode differ widely from one species to another. That would explain the huge differences in phenotypes that separate us from our rodent cousin or that most well-known of lab worms. Furthermore, in humans, a gene does not encode just one protein but seven on average. Hence the relevance of identifying all the proteins that the human body can produce, if we are to understand their interactions, their involvement in a given metabolic pathway or in the development of certain diseases. And this is where the Human Proteome Project (HPP) international consortium comes in. Launched 10 years ago, it enabled the publication last November of the first map of the human proteome, which is over 90% complete. "Each participating country synthesized internationally available information on a specific chromosome. France, for example, was in charge of chromosome 14, explains the researcher. With this team, federating scientists from four French platforms, we have managed to characterize over 300 missing proteins, i.e. those suspected but not proven to exist." How did they do it? To find out more, join us on a guided tour of the Protim platform.
Pineau, research director at the French Research Institute for Environmental and Occupational Health (Irset), is in charge of the Protim platform, which participated in the creation of the first map of the human proteome, shown on the screen. Each portion of the circle represents a chromosome. For each of them, the proportion of proteins already identified (18,357 in total) is shown in green and those only suspected to exist (1,421) are shown in other colors depending on the level of evidence. Thanks to the equipment at its disposal - here, in the right-hand photo, a MALDI mass spectrometry imaging device -, Protim enables Irset researchers to conduct their own research, collaborate with other teams, and offer services. It is thus integrated into the Grand Ouest interregional network of technology platforms in life sciences and the environment: Biogenouest.
The bottom-up approach, which starts from the most fundamental element and then expands the scale of observation, is the most common approach to identifying proteins. First step: the protein assay, carried out here by research engineer Blandine Guével, makes it possible to determine the total quantity of proteins in a sample. The method used, called "Micro BCA", results in the formation of a colored complex: the darker the wells of the microplate, the more proteins they contain. An essential count for preparing the quantity of biological material needed for the next step.
Second step: the digestion of proteins. These are not analyzed whole but digested with a cocktail of enzymes to obtain protein fragments called "peptides". These are soluble in the solution that will be injected into the mass spectrometer. Digestion is crucial to the preparation of samples: if it is incomplete, protein identification will be difficult.
Third step: fractionation of the peptides obtained after digestion, performed by electrophoresis. This technique allows the different peptides to migrate, under the effect of an electrical field, according to their mass - the heavier ones will migrate less readily. The objective? To check the quality of the digestion.
The final step is the analysis in the mass spectrometer, carried out here in the dust-free environment of a clean room by research engineer Régis Lavigne. This equipment makes it possible to measure the mass of each peptide resulting from the digestion of proteins. If certain masses correspond to the information present in the existing databases, researchers can reconstitute, like a puzzle, the entire protein, determine its amino acid sequence and thus identify it.
Slides for the mass spectrometer are prepared from frozen tissue, here from the epididymis, a tube connecting the testicle to the prostate. The tissues are first cut into very thin slices of 10 to 20 μm thickness by a device called a "microtome." The sample is then covered with matrix (yellow), a small organic molecule which, under the effect of the laser, will transfer electrons onto the proteins it contains and enable their detection.
The flagship of Protim's equipment, the MALDI mass spectrometry imaging device is capable of identifying proteins contained in a tissue, without digesting them first. This new approach makes it possible to study whole proteins, localize them in tissues, and follow their expression over time. On the right, this plate contains the samples to be irradiated in the spectrometer. Once the samples have been irradiated, and the various mass spectra generated, a software application makes it possible to reconstitute a "map" that locates several biological compounds already identified - proteins, peptides, sugars, lipids, metabolites... - and present simultaneously in a tissue. An open window, at time T, to their possible interactions!
The UV laser beam that will irradiate the tissue sample in the mass spectrometer is at the subject of the discussions between Pineau and Sarah Lennon, analytical chemist and researcher at Institut Pasteur in Paris. For each targeted area, called a "spot", a mass spectrum is generated, before moving on to a new spot.
* unit 1085 Inserm/Université de Rennes 1/EHESP School of Public Health, French Research Institute for Environmental and Occupational Health