Past and present
Mass spectrometry based proteomics has matured over the last decades and taken by now a strong position in the life sciences, but it clearly has not reached the required stage for large-scale biomedical and clinical applications in terms of comprehensiveness of analysis, robustness and availability. Global protein analysis poses a tough analytical challenge. The proteome is extremely multifaceted due to splicing and a plethora of co-occurring protein modifications, further amplified by the interconnectivity of proteins into complexes and signaling networks that are highly dynamic and divergent in time and space. Proteome analysis heavily relies on mass spectrometry (MS), and major progress in this technology have advanced the proteomics (and metabolomics) field. Beyond the omics-integration the proteome analysis by itself still needs to move much forward beyond the current state of the art. In the analysis of protein post-translational modifications, a wide field is still underexplored. While high-throughput qualitative and quantitative analysis of protein phosphorylation is now possible, partly through innovations introduced by the Netherlands Proteomics Centre, other PTMs such as ubiquitination, lipidation and glycosylation represent still much harder nuts to crack. New tools to start the enormous heterogeneity introduced by protein glycosylation, are very much needed, as it is well known that also aberrant glycosylation represents a hallmark of disease. Additionally, the structural organization of the proteome and how this changes upon external perturbations needs much further exploration, as this represents a missing link in translating quantitative proteome data with resulting phenotypes. Emerging methods such as protein cross-linking and native mass spectrometry can contribute to this understanding, especially in combination with more conventional method in structural biology, such as crystallography and electron microscopy, as proposed for instance in the KNAW program Bioscopy. An additional challenge is to integrate further proteome data with data generated at other levels, such as genomes, transcriptomes and metabolomes, in which bioinformatics will have a key role. Through a combination of developments in instrumentation, sample preparation and computational analysis, partly developed in the Netherlands, MS-based proteomics has matured and delivers now to many areas of biomedical and biological research. However, with this increasing need and impact, new questions in proteomics (and even X-omics) are becoming addressable. Here we seek to invest into this emerging next-generation of proteomics technology, focusing on high-end applications being widely accessible to top-researchers in the Netherlands and Europe.
Recent developments in the state-of-the-art
Proteomics focuses on system-wide analysis of proteins with the goal to understand their biological function in the context of all the other biomolecules present in the cells and/or tissue. For example, it allows studying the regulatory effect of protein interactions and modifications on cancer and autoimmune diseases. Moreover, as nearly all used drugs target proteins, proteomics has led to a better understanding of drug-treatments, in the context of the emerging view that such treatments should be personalized, avoiding unwanted side-effects. Proteomics has become a pivotal technology for research in the life sciences; it provides the crucial link between information presented by gene sequencing and the phenotype of the disease or other relevant studied biological process. Proteomics tools are nowadays used in a broad spectrum of applications, ranging from the detailed study of inter- and intra-cellular signaling, monitoring early diagnostic protein biomarkers of disease, to application in structural biology. During the last decade, the realization has emerged that successful proteomics requires an expensive and a constantly evolving infrastructure as well as a critical mass/size including well-trained high-level personnel.
Challenges to be addressed by the infrastructure
Over the next decade, both a consolidation of facilities for high-throughput quantitative proteomics, as well as investments in new proteomics technologies are foreseen. Next to a continuation of the current increasing demand of MS-based proteomics, focusing on proteins and their interactions, we aim to incorporate in the next phase dedicated infrastructures for glycoproteomics, which is a rapidly emerging field. Although we have entered this area already, we foresee its growing impact and relevance in the future. Next to dedicated instrumentation required to detangle glycan fine structures, we aim for comprehensive glycopeptide profiling methods with high level of annotations. This poses significant demands on instrumentation for fast sequencing/fragmentation and software for matching glycan and peptide sequences. In addition, methodologies for intact glycoprotein analysis will be further optimized. We would aim to add expertise in this emerging field into the consortium. Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. This requires the development of methods and instrumentation for phosphoproteomics, which are more sensitive, and have a higher throughput. The same holds for other modifications playing a regulatory role in the cellular context, such as Ubiquitination, OGlcNAc-ylation and methylation. These represent all areas that cannot be covered by DNA/RNA sequencing, adding relevant complementary data to such other –omics measurements. Another field of intense growth is the analysis of endogenous peptides, such as HLA peptides, neuropeptides and endogenous peptides present in body fluids such as plasma, milk and urine. The HLA peptides play a central role in for instance vaccines and in cancer immunotherapy. Such detailed analyses require new instrumentation and new sequencing methods, such as electron-transfer induced dissociation and UV-photodissociation. Another area of research that will prosper in the next phase is mass spectrometry based structural biology, using a combination of cross-linking mass spectrometry, native mass spectrometry of intact protein complexes, protein food printing and HD exchange MS. Structural Biology seeks to understand the relationships between structure and function of large protein complexes, and therein hybrid approaches are the future, for instance by coupling data gathered by cryo-EM microscopy or NMR, with MS-based data. These MS-based tools are of interest in fundamental structural biology, but also in characterization and quality control of biopharmaceutical products such as mAbs. We have already an established track-record in this area, but notice the rapidly growing demand for such facilities in the last few years. We should therefore invest more in dedicated infrastructures for MS-based structural biology and middle-down and top-down proteomics, including instruments equipped with alternative peptide sequencing methods (e.g. UVPD and EThcD) and instruments with larger mass ranges. We are working and collaborating on revolutionary approaches in proteomics, focusing currently on new instrumentation for nano mechanical MS mass spectrometry and position-sensitive imaging of protein complexes. These out-of-the-box new developments that we explore together with researchers from Caltech and Maastricht University, and technology companies such as Thermo Fisher Scientific and the Dutch SME MSVision illustrate our ambition to be at the front of future and emerging proteomics technologies.
Albert Heck is chair of Biomolecular Mass Spectrometry & Proteomics, affiliated to the Departments of Chemistry and Pharmaceutical Sciences of Utrecht University. The overarching theme of his research is to develop and implement mass spectrometric methods for the efficient and detailed characterization of proteins in relation to their biological function. Heck has pioneered the development of alternative peptide sequencing technologies. His group develops mass spectrometers dedicated to the analysis of intact proteins and protein complexes. As an inventor and innovator, he is well-recognized by academic and industry alike. Heck was scientific director of the Bijvoet School for Biomolecular Sciences and the Netherlands Proteomics Centre for more than a decade. He also provided a critical contribution to Proteins@Work.