MASSTRPLAN – MASS spectometry TRaining network for Protein Lipid adduct ANalysis
Project Description – Non-specialist
Diabetes and obesity are the cause of major health problems in modern society, with more than a quarter of the UK population now being classed as obese. These conditions are part of something called metabolic syndrome, a cluster of conditions that also includes high blood pressure, insulin resistance and high blood LDL cholesterol levels, all of which increase the risk of heart attack, stroke, and kidney diseases. These diseases are linked to increased inflammation, which occurs when the immune system becomes activated and produces lots of damaging compounds. Some of these damaging compounds attack molecules in the human body by a process that involves adding oxygen atoms (referred to as oxidation).
In situations where there is excess fats or “lipids” such as cholesterol and triglycerides circulating in the blood, the lipid molecules can be attacked and oxidized. These oxidized lipids become sticky (like old cooking oil) and react with the proteins and cells in the body, changing their function.
MASSTRPLAN is a European Network Project training 14 young researchers in 6 different countries. The focus of this training network is on the effects of the “sticky lipids” that occur in high-fat inflammatory conditions and are thought to contribute to the cellular malfunctions in cardiovascular disease and diabetes. The researchers are studying the proteins that are most susceptible to attack by sticky lipids, how this changes the function of these proteins, and why this makes cells behave in a way that causes disease.
As the sticky lipids and sticky-lipid-proteins are thought to be potential markers for early stages of disease, the project is also developing new methods of detecting them, mainly using an advanced technique called mass spectrometry that measures the mass of molecules, but also by making specific antibodies to be used in simple “point-of-care tests” for use in the patients’ homes or GPs surgeries. These would be similar in design to pregnancy test kits that can be purchased over the counter.
Project Description – Expert
Chronic inflammatory-based diseases such as diabetes, cardiovascular disease and cancer are major causes of mortality and morbidity in the EU and cost the economy dearly in health care and lost working time. Better understanding of the disease processes and biomarkers for earlier diagnosis are needed to improve treatment and outlook, which requires studies at a molecular level. The inflammatory component of these diseases leads to production of oxidizing, nitrating and halogenating compounds that cause oxidative modifications to a broad range of biomolecules. Phospholipid oxidation generates a wide variety of products including oxidised, nitrated and chlorinated lipids, many of which are strongly linked to aetiology of inflammatory diseases, although the mechanisms are not generally well understood. Electrophilic oxidized and nitrated lipid products can react with lysine, histidine and cysteine residues of proteins to form covalent adducts, a process called lipoxidation. Cysteine, methionine, tyrosine, tryptophan, proline, lysine and other residues in proteins can also be oxidatively modified directly. These oxidative post-translational modifications (oxPTMs) have many effects, including on enzyme activities, protein-protein interactions, and immune recognition, and are thus fundamental to regulating cell behaviour in physiology and pathophysiology. Both lipid oxidation and (lip)oxidation of proteins can disrupt cellular signalling and regulation, and there is strong evidence that they contribute to progression of cancer, atherosclerosis, neurodegenerative, metabolic and pulmonary disorders. Research interest in the combined lipid-protein-oxidation field has increased sharply in the last 5 years as demonstrated by PubMed searches, reflecting the growing awareness of its importance. However, methods for their detection and analysis are lagging behind.
Oxidized lipids and proteins are indicators of pathological oxidative stress, and have potential as diagnostic biomarkers for disease if precise and quantitative information can be obtained on the specific protein modified and type of oxidative modification. Advanced molecular and submolecular analysis is required to address this question, and the only technique able to distinguish different modifications and identify the proteins is mass spectrometry.
Introduction – Mass spectrometry (MS) for detecting oxidative modifications
Mass spectrometry is a fundamental analytical technique used in chemical and biochemical sciences, with extensive applications in Pharma and Biotechnology industries (Fig B1.1). MS technology has advanced enormously in the last 20 years, and sophisticated methodologies are available to help characterize structures and identify molecules. It has underpinned the explosion in OMICs approaches, particularly proteomics, glycomics and lipidomics, which are separate fields for analysis of proteins, sugars and lipids. These technologies are having a major impact on biomedical research and diagnostics, for example in the detection of cancer biomarkers, and are already revolutionizing services underlying medicine. However, approaches that integrate these “-omics” fields are rare. Moreover, the relative ease of automated analysis has led to a “push-button” approach, and many of the key skills in the basic science of LC and MS no longer form part of basic training, and while standard proteomics approaches to identify proteins and measure relative expression levels are routine, they are not well-adapted for analysis of oxidative and lipoxidative PTMs (oxPTMs). Specifically, the LC-MS methods and proteomic data handling algorithms, which were designed for protein identification, are not well suited for oxPTM detection, and result in many false positive and false negative identifications. Some PTM analysis, for example phosphorylation, has become more routine due to extensive research on enrichment and identification, but the analysis of heterogeneous PTMs caused by oxidation, nitration, glycoxidation and lipoxidation is not standard and continues to be very challenging. These oxPTMs are very heterogeneous: many hundreds of different ones are possible6. Their identification requires the development of sample handling methods that avoid adventitious atmospheric oxidations, and specially tailored instrument routines, often combined with chemical labelling and enrichment strategies and advanced chromatographic separations, and supported by extensive and complex manual data analysis.
Although some methods for limited oxPTMs are available, further multidisciplinary research is required to develop comprehensive methods for the majority of PTMs, especially lipoxidation, in order to provide information on specific oxPTMs, to understand their roles in inflammatory diseases and translate this information to biomedical and biotechnology applications. Currently available approaches are far from routine: young researchers are often only trained in the routine use of current omics methods and are unaware of the limitations of standard proteomics for identifying oxidative modifications.
Consequently, there is a key need for training in the application of advanced chromatography and mass spectrometry (MS) to oxidized protein and lipid analysis, and advanced data handling to detect and quantify oxPTMs. Although Europe has excellent expertise in general proteomics and mass spectrometry and some current ITNs involve the use of MS for limited aspects of their research, there are no PhD training programmes or ITNs focused on tackling the problems of identifying and quantifying oxidative modifications in inflammation by MS.
A cohort of early stage researchers with advanced instrument training in MS and analytical technologies to detect inflammatory damage will be a key resource that allows the EU to take the lead worldwide in this field, ahead of the US and Far East. The MASSTRPLAN ESRs will become the next generation of research leaders and industrial scientists, thus enabling expansion in this field and providing a highly competitive base. MASSTRPLAN will advance the MS methodology and knowledge on oxidative modifications in inflammatory disease, and train 14 ESRs in its use, thus filling the current gap in this training area. The methods developed have potential for broad translation to other fields, such as analytics for commercial protein production and biopharmaceuticals, a major industrial growth area in EU and worldwide. Each beneficiary contributes specific expertise to provide ESRs with a unique skills set (Fig B1.2).