Periodic Reporting for period 2 - MASSTRPLAN (MASS Spectrometry TRaining network for Protein Lipid adduct ANalysis) Reporting period: 2017-10-01 to 2019-09-30 Summary of the context and overall objectives of the project Many chronic, life-threatening diseases, e.g. diabetes and cancer, involve inflammation, which produces oxidizing compounds that damage cells and tissues. Oxidative damage to fats (lipids) makes them highly reactive or “sticky”; these can bind to proteins inducing further damage by altering the proteins’ structure and function, contributing to disease. This process is called lipoxidation. Being able to measure which proteins are affected by lipoxidation and how this alters their behaviour will improve our understanding of disease, leading to new diagnostic or therapeutic approaches. Understanding the consequences of lipoxidation and its link to diseases requires multidisciplinary approaches. The only method to characterize lipid oxidation and protein lipoxidation at a molecular level is mass spectrometry, but this is challenging, and few young researchers gain hands-on experience; industry particularly recognizes this shortage of scientists adequately trained in advanced analytical methods. The MASSTRPLAN project provided early stage researchers (ESRs) with a deep knowledge of the mass spectrometry of oxidized lipids and proteins combined with complementary techniques such as microscopy, flow cytometry & bioinformatics, and a broad perspective on disease mechanisms and diagnostic tools. These broadly skilled ESRs will support the translation of knowledge and techniques to industry and healthcare, ultimately generating outcomes that can benefit society. To achieve this, the MASSTRPLAN ITN brought together a multi-disciplinary network of 6 academic institutions, 2 hospitals and 2 commercial teams, and 14 other partners working in the fields of technology, pharmaceuticals, life sciences and learned societies. The main objectives of MASSTRPLAN were to:1. Train 14 ESRs in advanced and novel chromatography, mass spectrometry and complementary techniques including microscopy and bioinformatics, to detect challenging heterogeneous biomolecule modifications and determine their functional effects;2. Give ESRs a broad perspective on the relevance and mechanisms of oxidative modifications in pathophysiology and biotechnology; 3. Enable ESRs trained in technology development to engage effectively with the clinical sector; 4. Train ESRs in translational and development skills to produce new protocols, materials and commercializable diagnostic tools.The key outcomes of the project are the successful training of the ESRs and their introduction to the international scientific community (leading to high level employment), and extensive research outputs, already published in more than 50 articles. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far TRAINING ACTIVITIES. During the MASSTRPLAN project, the researchers benefitted from excellent and diverse training. They had extensive theoretical and practical training in mass spectrometry, microscopy and flow cytometry through webinars and network training events, including an industry-led training course hosted by SCIEX, ThermoFisher Scientific and Waters. They attended 8 network training events in 6 countries, including two summer schools, and heard lectures by many leaders in the field. The first summer school for MASSTRPLAN researchers was held at the Universidade de Aveiro (PT). The 2nd summer school organized by MASSTRPLAN was a FEBS Advanced Lecture Course cofunded by IUBMB and our partners SFRR-Europe and EuPA, with 80 participants. The ESRs also received tuition in soft skills such as scientific presentations, public engagement, and commercialization. A secure repository of all training materials was built for the researchers to access. A strength of the project has been the large number of secondments undertaken (on average 3 per researcher) that allowed them to experience diverse working environments and techniques in industry and academia.SCIENTIFIC PROGRESS AND DISSEMINATION. We accumulated a large amount of new scientific information, enhanced by extensive collaborative work between the teams and partners. The research was divided into four scientific work packages, each with a specific focus. In WP1 we developed new mass spectrometry-based methods for separating and identifying modified phospholipids, involving the use of molecular fragments as markers. We reported new evidence that modified lipid products attack specific proteins and found new markers to use in diagnostic tests. In WP2, we developed software to help to manage the large amounts of data generated by mass spectrometry, and find the modified molecules more efficiently. These bioinformatic tools are freely available. In WP3 we identified proteins susceptible to attack by reactive lipids and showed how this affects their structure and function, leading to changes in cell behaviour relating to disease. We also showed that obesity, cardiovascular diseases and cancers change the lipid content of cells and tissues, and modified lipids have specific inflammatory effects. Finally, the translational research in WP4 generated new antibodies to modified proteins and used them to make new assays and prototype kits to measure lipoxidized albumin, as well as providing optimised mass spectrometry protocols for the community.The achievements of the MASSTRPLAN network have been widely disseminated by a variety of complementary means. By November 2019, more than 50 published articles are in print, consisting of primary research articles in high impact factor journals, review articles summarizing key aspects of the field and recent advances, and published conference abstracts. We organized two Special Issues in our partner Elsevier’s journals Redox Biology and Free Radical Biology and Medicine. The research has been widely publicised by presentations at scientific conferences in Europe and the U.S. both by principal investigators and ESRs, and the latter have won several awards. MASSTRPLAN organized an independent, open conference entitled “Advances in the Study of Lipid and Protein Oxidation: From Methods to Targets”, which was attended by nearly 100 participants from 16 countries, as well as running the FEBS Advanced Lecture course on Redoxomics as described above. The researchers made presentations at local public events and schools, and organized an open public lecture entitled “We are what we eat”. We made a radio broadcast on this topic at the University of Gent, and 2 YouTube videos on the project. The ESRs managed the MASSTRPLAN website, set up a Facebook page (@masstrplan), LinkedIn account (https://www.linkedin.com/in/masstrplan-itn-934705131/) and Twitter account (@MSSTRPLAN2016), which are frequently used to disseminate news from the net Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In summary, MASSTRPLAN has advanced the fields of lipid oxidation and lipoxidation substantially beyond the state-of-the-art, providing new methods and tools for its detection, new understanding of its effects on cells and, in collaboration with clinical teams, its occurrence in disease. Through interaction with industrial partners, the new knowledge has been translated for future commercial benefit, including tools for bioinformatics and diagnostic assays. The advances in scientific knowledge and provision of highly trained researchers in the field is expected to lead to new approaches in the healthcare and industrial sectors in the next 5 years, with a concomitant longer term benefit to the wider society.