SPIDoc’s DN stems from the need to prepare for emerging viruses enabling a fast response, as became evident lately. Basically, better knowledge of the dynamic nature of viral structures is required and fundamental to combat with appropriate treatments. In this perspective, several technologies were developed for structural biology, including a cutting-edge approach combining mass spectrometry (MS) and X-rays for single particle imaging (SPI) in MS SPIDOC. Despite these advances, a major bottleneck that the scientific community will face in coming years will be the lack of expertise with a broad knowledge from theory, instrument development and application relevant to structural biology with MS. SPIDoc’s therefore aims to train the next generation of scientists with such expertise by a dedicated intersectoral training programme. SPIDoc’s projects are highly interlinked and the proposed training programme aims to provide the doctoral candidates with the basic and advanced knowledge to build a common interdisciplinary language, to work in teams and to acquire deep knowledge by a combination of lectures and practical sessions. An important aspect will be given to data management and the establishment of currently lacking standards for native MS metadata. The research training is complemented by transferrable skills courses, setting the basis for progressing through the doctoral studies as well as for future career progression. Within these modules, training on equal opportunities and unconscious bias are included, shaping the future generation towards creating fair workplaces. Special attention will be paid to green-practice measures from the everyday practice in laboratories and offices up to the institutional level. By including developments from computational theory to instrumentation, the SPIDoc’s beneficiaries and associated partners will closely work together to advance cutting-edge technologies and apply them towards fast response.

Mass spectrometry has become a multi-billion dollar market word-wide, because it allows one to quantitatively assess the molecular content of a sample and to retrieve molecular structure information. SUPERMAMA now aims at breaking new scientific grounds for new technologies that shall boost the capabilities of mass spectrometry as well as of optical spectroscopy. Here we specifically target singly charged and neutral high-mass proteins. SUPERMAMA will develop, test and combine the first integrated superconducting nanowire array (SNWA) with advanced cryogenic onboard electronics in a largely re-modelled ESI-TOF-machine. The efficient detection of massive biomolecules at low kinetic energy will be an important first milestone for mass spectrometry. The development of a new generation of photocleavable tags shall allow the preparation of neutral protein beams from mass-selected ions in focused transverse high-power laser fields. Photo-cleavage post-ionization of tagged proteins shall also be studied as a generic tool to decouple the volatilization from the charging process. This will enable the combination of a systematic analysis of neutral proteins in the gas phase with subsequent mass spectroscopy. The combination of all techniques shall open new avenues for few-photon calorimetry and single-photon recoil spectroscopy. The calorimetry studies will explore the sensitivity of SNWA detection to molecular heat. Future experiments will study the shift of molecular matter-wave interference fringes caused by the recoil of a single photon. Two industrial and three academic research teams represent a highly interdisciplinary consortium of experts from mass spectrometry, superconductor technology, integrated electronic engineering, synthetic chemistry, as well as molecular beam physics and quantum optics who work together in towards their joint goal to advance mass spectrometry and optical spectroscopy in a domain that has remained unexplored so far.

The European XFEL has just entered user operation. With its unparalleled peak brilliance and repetition rate, European XFEL has the potential to further applications in single particle imaging (SPI), thus far limited to large viral particles at X-ray Free-Electron Lasers (XFEL). SPI will allow imaging protein complexes without the need for crystallization. This eventually renders transient conformational states accessible for high resolution structural studies yielding molecular movies of biomolecular machines. A major bottleneck is the wealth of data required to reconstruct a single structure leading to long processing times. This is currently also a problem in electron microscopy (EM). MS SPIDOC will overcome this data challenge by developing a native mass spectrometry (MS) system for sample delivery, named X-MS-I. It will provide mass and conformation selected biomolecules, which are oriented along their dipole axis upon imaging. This will enable structural reconstruction from much smaller datasets speeding up the analysis time tremendously. Moreover, the system features low sample consumption and a controlled low background easing pattern identification. The main objectives of the project are: • Deliver mass and conformation separated biomolecules for SPI. • Orient proteins for SPI. • Image protein complex unfolding • Exploit potential of protein orientation for other applications The MS SPIDOC consortium combines internationally leading expertise in different fields relevant to the project: Instrument design and development, computer simulations as well as working with biomolecules in the gas phase and on SPI are combined to implement the novel sample environment at the next generation XFEL facility. New components and methods will be opened to the market and thereby strengthen the European Research Area (ERA) and industry. This early stage high-risk project will give rise to a new technology with major impact on how to derive structures of biomolecules.

One of the major challenges of modern medicine is to understand how the human organism defends itself against invasions and diseases. The biggest mystery is the human immune system, and understanding this ultimately requires knowledge of the sequence repertoire of human antibodies and their respective antigens. The purpose of the TopSpec project is to be the first in the world to solve this challenge, opening up opportunities in medical research and drug development that are today only dreamt about. We will create a breakthrough technology that will revolutionize academic, clinical and industrial proteomics and dramatically advance the development of new generation antibody- and protein-based therapeutics. Antibodies represent the most sophisticated line of natural defense against disease. Knowing exactly which antibodies are produced in response to a particular disease enables us not only to better understand the cause of the disease but also to provide new-generation cures in the form of personalized therapeutic antibodies. The limiting factor for this to truly be achieved is to find a way to analyze and sequence large molecules in the gas phase, and this represents a formidable challenge. The TopSpec project will develop ground-breaking TOP-down tandem mass SPECtrometry (MS/MS) approaches based on novel radical gas-phase ion-electron and ion-atom reactions, and implement them on a unique, hyphenated, ultrahigh-resolution MS platform. Another “killer innovation” is the ability to greatly simplify MS/MS spectra of large molecules by adding another dimension of separation – collisional cross-sections of fragment ions using two parallel approaches. TopSpec will be the first project to implement de-convolution of massively overlapping isotopic clusters, solving one of the greatest challenges in top-down MS of large molecules.