Establishment and Evaluation of Affinity Reagents for Mass Spectrometry Based Proteomic Studies on Lysine Methylation

Abstract

Methylation of lysine (Kme) is known to regulate several important functions, altering gene expression and cell cycle progression. However, mass spectrometry based proteomic analysis of the modification has not been possible due to the lack of efficient purification strategies to enrich modified peptides for analysis. In this study, we identified and evaluated several in-house and commercial candidate Kme binders targeting the three states of methylated lysine, and proposed a novel purification strategy based on immunoprecipitation and magnetic beads. Binders that discriminate between the three states with high selectivity were identified, and the viability of different enrichment strategies based on immunoprecipitation with Protein A-coated magnetic beads and sepharose resin was evaluated. Additionally, a protocol for preparation of input material for these was established. In summary, the results are a significant step towards an operational affinity strategy for studies on lysine methylation proteomics, which would unlock vast possibilities for research on the role of Kme in many common disease states.

Popular Abstract

In 2020, the World Health Organization estimates that almost 10 million people will die from cancer worldwide, and in Sweden it accounts for 1 out of 4 deaths. The treatment for different cancers has historically been unspecific, but the many discoveries and new drugs developed in the last few decades have increased the survival rate significantly. Nevertheless, cancer is still the second most common cause of death worldwide, and further improvements in diagnostics and treatments are critical. Lysine methylation is a protein modification that could be important in diagnostics and treatment of cancer, but it is currently hard to study. In this project, we aimed to establish a new method to study the modification using binders attached to magnetic beads, and made progress that will be useful when trying to analyse lysine methylation in the future.

Almost all diseases are caused by proteins not functioning correctly. Proteins are perhaps most known for keeping our body strong and being a vital part of our diet, but they are involved in everything happening within our body. For instance, the protein haemoglobin in our blood transports oxygen from our lungs to the rest of our body. Proteins are made by the translation of our genetic code, DNA, into a long chain of molecules. The genes in our cells code for 20,000 proteins. However, proteins are not static but dynamic: to every protein, small modifications can be attached. This means that the number of modified protein variants is close to 1 million. When a modification is added to a protein, it changes its function. For instance, when some groups are added to a protein, they can cause it to become more active. Proteins can also be switched off entirely, or change their function, when a modification is attached.

Lysine methylation, the subject of this study, is an important and well-studied modification. It occurs when methyl groups, essentially small carbon groups, are added to a specific part of the protein, lysine. Depending on how many of these groups are attached, the function of a protein can change. For instance, the addition of another methyl group can lead to a change in the
cell cycle, which determines how fast cells can multiply. Cancers can exploit this to spread faster throughout the body. Needless to say, understanding lysine methylation better could lead to the development of better medicinal targets for several diseases. However, it is not easy to study the modification. It is extremely small and very scarce compared to other modifications. Because of this, to be able to see how it impacts our proteins and how it can have a role in disease development, very good methods are an essential part.

The aim of this study was to find a good way to study lysine methylation in all proteins. The single most important factor in this is finding binders, called antibodies, that can detect the modification. In this study, we evaluated eight different binders. We found that four of the antibodies could distinguish between the different types of the modification very well. After this, we grew cells and extracted their proteins with the modification attached. For detection and analysis of the modification, the proteins were
broken into smaller fragments called peptides. Then, we attached the discovered binders to magnetic beads. The idea with this is that magnetic beads can be added to a mixture where the antibodies can catch the desired modified peptides, and then be removed with a normal magnet from the peptides that we don’t want. This procedure is visualized in Figure 1. We successfully bound the antibodies to the magnetic beads, and they did not come off during the entire process.

In the end, we did not manage to catch the desired peptides, because the antibodies changed when they were attached to the beads. However, as this was a short term project, there are still many parts of the process to optimize, and after this, with what has been discovered now, it is not unlikely that the method we used will function well. In summary, the discovery is a significant step on the way towards analysing what lysine methylation does in detail, and how it may help us save lives by being a key part of future cancer treatments.

• author: Hermansson, Albin
• supervisor: Magnus Jakobsson
• organization: Department of Immunotechnology
• course: KIMM05 20201
• year: 2020
• type: H2 - Master's Degree (Two Years)
• subject: Medicine and Health Sciences
• language: English
• id: 9023835
• date added to LUP: 2020-08-13 09:43:48
• date last changed: 2020-08-13 09:43:48