Do medicines alter our proteome?

Although physicians administer and prescribe drugs to patients due to their desired therapeutic effects, their use is also associated with potentially adverse side effects, which are not always well understood, particularly in the early stages of treatment. Professor Piotr Suder’s team from the Department of Analytical Chemistry and Biochemistry at the AGH University of Krakow is conducting research that helps fill this gap: it enables a detailed understanding of the effect of various substances on the proteome, i.e. the protein composition of human and animal tissues.

Changes in the proteomic composition are a natural manifestation of how organs function under the influence of various factors. These changes may reveal the biological mechanisms underlying the therapeutic effects of drugs, highlight the initial signs of various diseases or disorders, or indicate an early stage of tissue or organ damage. Medicines are among the factors that influence the proteome; changes in the proteome may provide insights into the benefits and risks of their use in unprecedented ways. From this perspective, proteomic data can help doctors tailor treatment to the current state and individual needs of the patient with greater accuracy.

However, researchers emphasise that such detailed analyses are only possible thanks to technological advances made over the last several years, and although there had already been attempts to carry out such research, the current level of precision was not available to scientists until very recently.

Thanks to specialist equipment, the team of researchers from AGH University is able to conduct detailed, simultaneous analyses of concentration changes in several thousand proteins within a single biological sample, which allows them to draw conclusions about changes in the functioning of studied tissues.

Based on these results, it is possible to rule out one of the hypothetical mechanisms of action of the drug, redesign its structure to reduce toxicity, or combine it with another drug that will enhance its effects whilst remaining safe. Sometimes no changes occur, or they occur only in, for example, tumour tissue, without any changes in normal cells. This would also be a valuable piece of information on the effects of the substance.

The equipment used by researchers at AGH University includes high-performance liquid chromatography with capillary flow rates, i.e. flow rates of hundreds of nanolitres per minute (1 millilitre of the solutions used allows for over two days of operation). It is connected to a mass spectrometer, a high-resolution instrument capable of detecting the substances under investigation with femtomolar sensitivity. These are rather abstract quantities: if you were to add a teaspoon of sugar (3 grams to around 3 million litres of water) to an Olympic-sized swimming pool and, after stirring, take a 1 millilitre sample, the amount of sugar in that volume would still be over one thousand times greater than what the scientists are able to detect. The technology applied by the research team is widely recognised around the world as it is extremely sensitive, enabling the detection of many thousands of substances in a single analysis. It allows for the analysis of a wide range of samples and is also relatively quick, as a single analysis can be completed in just 90 minutes. In such a sample, scientists can observe around five to six thousand proteins, sometimes even more. The less advanced methods used previously, such as two-dimensional electrophoresis, allowed the analysis of two to three thousand of the most concentrated proteins and the process took several weeks.

Morphine alters the proteome of the kidneys and liver

One of the drugs whose effects were analysed in detail by the AGH University team using the abovementioned methods was morphine, which served as a model substance for the entire group of opioids (strong painkillers). Based on this research, Małgorzata Hopciaś completed a master’s thesis that was later recognised as the best theoretical thesis at AGH University in the 2024/25 academic year.

Like other opioids, morphine is used as a powerful painkiller when other methods of pain relief fail. It is considered relatively safe, but its long-term use may have adverse effects on certain organs, such as the liver and kidneys. Unfortunately, it is also sometimes used as a narcotic substance that is highly addictive. Understanding the type of tissue damage caused by morphine, and when serious or irreversible changes begin to occur, may allow a safe dose to be personalised to the patient, and to find better, more effective ways of alleviating side effects or preventing them from occurring at all.

The study was conducted using animal tissue and showed that morphine does indeed cause changes in the proteome of the liver and kidneys. The processes taking place in the liver have been found to be function-specific; in other words, they are linked to the liver’s role in metabolising various types of substances, that is, breaking them down to reduce their toxicity and facilitate their excretion. Following the administration of morphine, there was an increase in the levels of various enzymes responsible for its breakdown and conversion. Therefore, it can be concluded that the dose-dependent changes in the liver proteome did not indicate the occurrence of a process harmful to the function of this organ. The changes observed in the kidneys proved to be more concerning as they involved a disruption in the activity of certain proteins. The researchers believe that this observation may indicate the presence of early-stage kidney damage in the studied model.

As emphasised, morphine is a relatively safe drug, particularly for people with no history of chronic liver or kidney disease, in whom it is used in controlled, low doses for a short period of time. However, the correlations identified may be of significant importance for the treatment of older people or those with underlying conditions who have previously experienced kidney problems. Perhaps the use of opioids in these patients should be subject to closer monitoring, which would allow for intervention before the drug causes irreversible damage to the body.

It is also worth noting that, although animal studies may point to potential risks, we should treat them merely as a rough guide and must not uncritically extrapolate their findings to the effects of opioids on humans. Be that as it may, the findings obtained so far suggest which processes are worth focusing on and point the way for further research.

Sample preparation is critical to obtaining a reliable result

To begin proteomic research, it is necessary to obtain material that is representative of the model under study. AGH University does not keep animals from which tissue could be obtained; therefore, in order to secure such tissue, our researchers collaborate with other research centres. Professor Suder’s team collaborates with, among others, the Medical University of Lublin. The isolated tissues from animal models supplied for research are often fragments of organs such as the brain, liver, or kidneys.

Depending on the analytical method used, the collected samples must be prepared adequately. The procedure adopted by the AGH University team calls for the samples to be homogenised under rigorously controlled conditions in a carefully formulated solution. As a result, a homogeneous solution is obtained, containing all the proteins from the tissue under examination. To use the method developed by the researchers, it is also necessary to break down the proteins, i.e. to isolate peptides from them. This process occurs when an enzyme called trypsin (an enzyme known for its high efficiency in various ‘harsh’ environments, such as our digestive system) is added to the sample. Only a portion collected from such a sample is placed in an appropriate solution and subsequently analysed using high-performance liquid chromatography coupled and a mass spectrometer.

The researchers receive various types of samples, depending on the objectives of the research plan. There usually need to be at least two groups – one comprising individuals that have been exposed to a particular factor, such as a stressor or a substance whose effects are being studied, and the other comprising ‘control’ individuals that have not been exposed to these factors. By comparing the results of both groups, the academics can identify certain correlations and determine whether the factor under investigation causes differences in the proteomic composition of the samples.

Does altering protein composition help alleviate PTSD?

Other proteomic studies carried out by the researchers focused on post-traumatic stress disorder, or PTSD, and experimental drugs used in research into alleviating its symptoms. PTSD is a mental health condition that can be caused by a traumatic event, such as an accident or an experience of violence. It manifests itself in intrusive memories of the event, nightmares related to the past event, and severe anxiety, which usually significantly impair daily functioning.
The studies had two components: firstly, a comparison of the proteome composition of specific brain structures in healthy and affected individuals in a rat model of PTSD; and secondly, an assessment of the effects of an experimental drug. The researchers focused on two brain structures: the hippocampus and the amygdala. The former is responsible for consolidating memory traces, whilst the latter is involved in the experience of fear or terror; thus, both play a significant role in the development of PTSD.

The studies were carried out in collaboration with the Medical University of Lublin. In line with a relevant authorisation for animal testing, the pharmacologists subjected the rats to behavioural tests after inducing PTSD in them and then collected tissue samples for analysis. The main objective of the AGH University team was to determine whether the occurrence of PTSD is associated with changes in the proteome of specific brain structures, and to investigate whether the administration of medication might influence these changes. Such observations would suggest that the beneficial effects of drugs stem from their impact on the proteome of one of the brain’s structures. Preliminary results did not confirm any changes in the hippocampus, but clear differences were observed in the amygdalae, and these were partially reversible with the use of drugs. The animals also showed a significant improvement in performance following administration of the test drug in a selected behavioural test, which correlated with the proteomic analysis. On this basis, the researchers speculate that the drug may work by reversing the observed changes in the amygdala’s proteome, thereby inhibiting the experience of fear and mitigating the effects of a stressor.

Toxins may be less harmful

Yet another related research project was carried out in collaboration with a team of animal scientists from the West Pomeranian University of Technology in Szczecin. Researchers there are studying the impact of aflatoxins on livestock, such as pigs.
Although the presence of such toxins can have a negative effect on livestock farming, it is difficult to eliminate them entirely, as they generally enter the animals’ systems through their feed. It is difficult to subject cereals intended for animal feed to the same level of scrutiny as those intended for human consumption given their vast amounts. For this reason, other measures are needed to prevent the adverse effects of taking even small doses of aflatoxins.
Researchers at the West Pomeranian University of Technology administer various plant extracts with active substances to animals – substances that may have a protective effect or improve the metabolism of toxins – and then provide researchers at AGH University with samples of the livers and kidneys of the animals undergoing treatment. The team’s analytical work is still in its early stages, but the results are promising and have pointed the researchers in a direction that deserves further investigation.

Proteomic analyses have a bright future

At present, the AGH University scholars are consulting with a representative from Mayo Clinic (Rochester, MN, USA), a world leader in advanced medical care, clinical research and education. The aim is to optimise the technical specifications of the equipment available at the university, so as to improve its analytical performance to a significantly higher level than that achieved in routine testing. This would make it possible to carry out analyses using even smaller samples, with greater sensitivity and fewer measurement errors, which is currently a global trend in advanced biochemical analysis using nanoLC-MS/MS systems.
Proteome mapping is still largely experimental, meaning it is not yet widely used in medicine. However, doctors are beginning to recognise its potential in hospitals, in the diagnosis and monitoring of patients’ health, for example as biomarkers for various medical conditions. As with other medications or treatments, its introduction is time-consuming and can take several years before it becomes standard practice in hospitals and surgeries. Nonetheless, current academic research is already providing information with potential value for healthcare professionals, patients, and the pharmaceutical industry.